Essential fungal polynucleotides, polypeptides, and methods of use

ABSTRACT

The presnt invention provides essential fungal polynucleotides and their encoded polypeptides, homologues thereof and their uses. Additionally, the invention provides methods for the identification of essential polynucleotides and fungal strains which may be used for drug screening.

This application claims benefit to provisional application U.S. Ser. No.60/376,022 filed Apr. 26, 2002, under 35 U.S.C. 119(e). The entireteachings of the referenced application are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention provides essential fungal polynucleotides andtheir encoded polypeptides, homologues thereof and uses thereof.Additionally, the invention provides methods for the identification ofessential polynucleotides and fungal strains which may be used for drugscreening.

BACKGROUND OF THE INVENTION

As the number of severe fungal infections continues to rise, the needfor a broad spectrum antifungal agent becomes more urgent. The rise infungal infections is primarily due to the increasing number ofimmuno-compromised patients as a result of medical advances(transplantation and chemotherapy) and as a result of the increasingpopulation of AIDS patients.

Although fluconazole has been an effective drug against fungal pathogensfor a number of years, resistance is increasing. Alternatives such asamphotericin B have serious drawbacks, including such side effects asnephrotoxicity and severe discomfort. Few new antifungals are on thehorizon, and more knowledge about the pathogenicity of fungi as well asabout their general biology is crucial if new drug targets are to beidentified.

More than 80% of fungal infections in immuno-compromised patients arecaused by Candida species. Cryptococcosis is the second most prevalentfungal infection in AIDS patients following candidiasis. Aspergillosisis responsible for at least 30% of the infections in cancer and organtransplant patients and has a high mortality rate.

In order to discover new drugs to combat fungal infections, compoundsare often tested for their effects on particular, suitablepolynucleotides and polynucleotide products. Suitable polynucleotidesare generally those which are found to be essential to the viability ofthe pathogen. This determination of essentiality presents substantialobstacles to the identification of appropriate targets for drugscreening. These obstacles are especially pronounced in diploidorganisms, such as Candida albicans.

A central technique used to investigate the role of a Candida albicanspolynucleotide is to study the phenotype of a cell in which both copiesof the polynucleotide have been deleted. Two popular polynucleotidedeletion protocols have been reported. The first and most often usedmethod, the ‘urablaster’ method, requires construction of a disruptioncassette consisting of a selectable marker (URA and hisG flankingsequences) and sequences of the polynucleotide to be disrupted that arepositioned at the 5′ and 3′ ends of the hisG-URA3-hisG cassette. A ura3−strain is then transformed and grown on minimal medium lacking uridine.ura3+ clones are isolated and transferred to a medium containing5-fluoroortic acid, which selects for strains that have a ura3−genotype. These arise spontaneously as a subpopulation of the originaltransformed cells which will have undergone a recombinational event thatretains one of the hisG sequences in the disrupted allele. Theheterozygote ura3− strains can then be used in a second transformationevent using the same cassette to disrupt the second allele resulting ina homozygous deletion strain.

A second type of popular method utilizes a PCR-based polynucleotidedisruption strategy and multiple markers to construct homozygous mutants(Wilson et al J. Bacteriol. 181: 1868-74 (1999)). Although more rapidthan the urablaster method, both methods have limitations. Since ahomozygous deletion strain which lacks both essential polynucleotidecopies would not be viable, such results are not an unequivocalexplanation establishing the essential nature of the targetpolynucleotide because alternative explanations, including poor growthof a viable mutant strain, may be as likely a reason as essentiality forthe negative results obtained.

Essential polynucleotides may also be identified using induciblepromoter-regulated constructs to modify expression of the secondpolynucleotide copy, rather than completely inactivating it. With thesemethods, one polynucleotide copy is disrupted and the second copy isonly expressed under certain conditions. The essentiality of thepolynucleotide can be investigated since the fungal strain will only beviable under conditions in which the promoter is switched on (See, forexample Nakayama et al. (Infection and Immunity 68: 6712-6719, (2000),and WO 01/60975).

One reportedly effective technique involves the use of the C. albicansMET3 promoter (Care et al., Molecular Microbiology 34 792-798 (1999)).The activity of the promoter is inhibited by methionine and/or cysteineand completely inactivated with both amino acids. Although the MET3promoter is not the only regulated promoter to be characterized in C.albicans, one advantage of this promoter is that it is controlled by theaddition of amino acids to the growth medium rather than a switch incarbon source. Switching carbon sources is likely to cause a biggerdisturbance to cell physiology than adding amino acids (Care et al).However, the method developed for use of the MET3 promoter by Care etal. is cumbersome since it requires subcloning a portion of thepolynucleotide under evaluation.

Additional methodologies enabling the identification of essentialpolynucleotides for drug screening which are both easy to use and giverapid results are needed, particularly for C. albicans. The C. albicansgenome-sequencing project has recently begun and novel polynucleotidesare being identified. There should be increasing demand to assessessential polynucleotide function and methodologies particularlyamenable to high throughput screening will be useful.

Furthermore, although the identification of novel polynucleotides asessential in C. albicans is of value, the determination of essentialityfor known polynucleotide sequences in C. albicans or other fungalgenomes is also highly desirable since such polynucleotides will add tothe set of drug targets. Furthermore, the use of C. albicans essentialpolynucleotides for drug screening for which orthologs in otherpathogenic fungi are identified may result in the discovery of drugseffective in fighting infections from a variety of pathogens.

SUMMARY OF THE INVENTION

The invention provides a nucleic acid molecule including nucleotidesequences that hybridize under stringent conditions to a second nucleicacid molecule having a nucleotide sequence that encodes an essentialpolypeptide having an amino acid sequence selected from the groupconsisting of one of SEQ ID NO: 12 to 22.

The invention also provides a substantially pure oligonucleotide, saidoligonucleotide comprising a region of nucleotide sequence capable ofhybridizing under highly stringent conditions to at least about 12consecutive nucleotides of one of SEQ ID NO: 1 to SEQ ID NO: 11.

The invention also provides a polynucleotide comprising the nucleotidesequence of a member of the group consisting of SEQ ID NO: 1 to SEQ IDNO: 11, or the nucleotide sequence included in the deposited clone.

The invention also provides a recombinant DNA molecule comprising theisolated nucleic acid molecule comprising a nucleotide sequence of amember of the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11, or thenucleotide sequence included in the deposited clone.

The invention also provides a recombinant DNA molecule comprising anucleotide sequence of a member of the group consisting of SEQ ID NO: 1to SEQ ID NO: 11, or the nucleotide sequence included in the depositedclone, wherein said polynucleotide is operably linked to one or moreregulatory sequences.

The invention also provides an expression vector comprising an isolatedpolynucleotide comprising a nucleotide sequence of a member of the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 11.

The invention also provides a host cell transformed to contain anexpression vector comprising an isolated polynucleotide comprising anucleotide sequence of a member of the group consisting of SEQ ID NO: 1to SEQ ID NO: 11, wherein said host cell is either a prokaryote or aeukaryote.

The invention also provides a substantially pure oligonucleotide, saidoligonucleotide comprising a region of nucleotide sequence capable ofhybridizing under highly stringent conditions to at least about 12consecutive nucleotides of one of SEQ ID NO: 1 to SEQ ID NO: 11.

The invention also provides a substantially pure oligonucleotide, saidoligonucleotide comprising a region of nucleotide sequence capable ofhybridizing under highly stringent conditions to at least about 12consecutive nucleotides of one of SEQ ID NO: 1 to SEQ ID NO: 11, whereinsaid oligonucleotide further comprises a detectable label attachedthereto.

The invention also provides a An isolated nucleic acid molecule obtainedfrom an organism other than Candida albicans or Saccromyces cervisiaecomprising a nucleotide sequence having at least 30% identity to asequence selected from the group consisting of SEQ ID NO: 1 to 11.

The invention also provides a method for producing a polypeptidecomprising the step of culturing a host cell transformed with thenucleic acid molecule comprising a nucleotide sequence of a member ofthe group consisting of SEQ ID NO: 1 to SEQ ID NO: 11 under conditionsin which the protein encoded by said nucleic acid molecule is expressed.

The invention also provides a polynucleotide that encodes a full lengthprotein of a member of the group consisting of SEQ ID NO: 12 to 22, orthe encoded sequence included in the deposited clone.

The invention also provides an isolated nucleic acid molecule obtainedfrom an organism other than Candida albicans or Saccromyces cervisiaeincluding a nucleotide sequence having at least 30% identity to asequence selected from the group consisting of SEQ ID NO: 1 to 11,wherein said identity is determined using the CLUSTALW algorithm withdefault parameters.

The invention also provides an isolated nucleic acid molecule obtainedfrom an organism other than Candida albicans or Saccromyces cervisiaeincluding a nucleotide sequence having at least 30% identity to asequence selected from the group consisting of SEQ ID NO: 1 to 11,wherein said identity is determined using the CLUSTALW algorithm withdefault parameters, wherein said organism is selected from the groupconsisting of Absidia corymbigera, Aspergillus flavis, Aspergillusfumigatus, Aspergillus niger, Botrytis cinerea, Candida dublinensis,Candida glabrata, Candida krusei, Candia parapsilopsis, Candiatropicalis, Coccidioides immitis, Cryptococcus neoformans, Erysiphegraminis, Exophalia dermatiditis, Fusarium osysproum, Histoplasmacapsulatum, Magnaporthe grisea, Mucor rouxii, Pneumocystis carinii,Puccinia graminis, Puccinia recodita, Rhizomucor pusillus, Pucciniastriiformis,, Rhizopus arrhizus, Septoria avenae, Septoria nodorum,Septoria triticii, Tilletia controversa, Tilletia tritici, Trichospoonbeigelii and Ustilago maydis.

The invention also provides an isolated nucleic acid molecule obtainedfrom an organism other than Candida albicans or Saccromyces cervisiaeincluding a nucleotide sequence having at least 30% identity to asequence selected from the group consisting of SEQ ID NO: 1 to 11,wherein said identity is determined using the CLUSTALW algorithm withdefault parameters, wherein said organism is Aspergillus fumigatus.

Additionally provided is an isolated nucleic acid molecule having afragment of at least 10 consecutive nucleotides of one of SEQ ID NO 1 to11.

The invention provides a substantially purified polypeptide including anamino acid sequence selected from the group consisting of one of SEQ IDNO: 12 to SEQ ID NO: 22 or one of SEQ ID NO:48 to one of SEQ ID NO: 73.

Furthermore, the invention provides a strain of Candida albicans whereina first copy of a polynucleotide having a nucleotide sequence selectedfrom the group consisting of one of SEQ ID NO 1 to 11 is inactive and asecond copy of the polynucleotide is under the control of a regulatablepromoter.

Furthermore, the invention provides a strain of Candida albicans whereina first copy of a polynucleotide having a nucleotide sequence selectedfrom the group consisting of one of SEQ ID NO 1 to 11 is inactive and asecond copy of the polynucleotide is under the control of a regulatablepromoter, wherein said regulatable promoter is MET3.

The invention also provides a strain of Candida albicans comprising anucleic acid molecule having a nucleotide sequence selected from one ofSEQ ID NO: 1 to 11 under the control of a regulatable promoter.

Additionally provided herein is an isolated nucleic acid molecule havinga nucleotide sequence encoding a polypeptide required for growth ofCandida albicans, wherein said polypeptide includes an amino acidsequence of one of SEQ ID NO: 12 to 22.

The invention also provides a method for identifying essentialpolynucleotides in diploid fungal cells, said method having the stepsof:

(a) inactivating a first copy of a polynucleotide in diploid fungalcells by recombination using a PCR-based polynucleotide disruptioncassette, thereby providing heterozygous diploid fungal cells;

(b) modifying a second copy of the polynucleotide in the heterozygousdiploid fungal cells by recombination using a PCR-based promoterswapping cassette including a nucleotide sequence encoding a regulatablepromoter, such that expression of the second copy of the polynucleotideis regulated by said promoter;

(c) culturing said cells with a promoter suppressor; and

(d) assessing growth of said cultured cells in comparison to controlcells.

The invention also provides a method for identifying essentialpolynucleotides in diploid fungal cells, said method having the stepsof:

(a) inactivating a first copy of a polynucleotide in diploid fungalcells by recombination using a PCR-based polynucleotide disruptioncassette, thereby providing heterozygous diploid fungal cells;

(b) modifying a second copy of the polynucleotide in the heterozygousdiploid fungal cells by recombination using a PCR-based promoterswapping cassette including a nucleotide sequence encoding a regulatablepromoter, such that expression of the second copy of the polynucleotideis regulated by said promoter;

(c) culturing said cells with a promoter suppressor; and

(d) assessing growth of said cultured cells in comparison to controlcells, wherein said diploid fungal cells are Candida albicans cells,wherein said polynucleotide is a conserved gene, wherein said promoteris a MET3 promoter, wherein said promoter suppressor is methionine,wherein said promoter suppressor is cysteine, and/or wherein saidpromoter suppressor is both cysteine and methionine.

Additionally provided herein is a method for inducing drughypersensitivity in diploid fungal cells, said method including thesteps of:

(a) inactivating a first copy of an essential polynucleotide in diploidfungal cells by recombination using a PCR-based polynucleotidedisruption cassette, thereby providing heterozygous diploid fungalcells;

(b) modifying the second copy of the polynucleotide in the heterozygousdiploid fungal cells by recombination using a PCR-based promoterswapping cassette including a nucleotide sequence encoding a regulatablepromoter, such that expression of the second copy of the polynucleotideis regulated by said promoter;

(c) culturing said cells with a promoter suppressor and with a drug; and

(d) comparing the effects of said drug on the growth of said cells incomparison to control cells.

Additionally provided herein is a method for inducing drughypersensitivity in diploid fungal cells, said method including thesteps of:

(a) inactivating a first copy of an essential polynucleotide in diploidfungal cells by recombination using a PCR-based polynucleotidedisruption cassette, thereby providing heterozygous diploid fungalcells;

(b) modifying the second copy of the polynucleotide in the heterozygousdiploid fungal cells by recombination using a PCR-based promoterswapping cassette including a nucleotide sequence encoding a regulatablepromoter, such that expression of the second copy of the polynucleotideis regulated by said promoter;

(c) culturing said cells with a promoter suppressor and with a drug; and

(d) comparing the effects of said drug on the growth of said cells incomparison to control cells,

wherein said diploid fungal cells are Candida albicans cells, whereinsaid polynucleotide is a conserved gene, wherein said promoter is a MET3promoter, wherein said promoter suppressor is methionine, wherein saidpromoter suppressor is cysteine, and/or wherein said promoter suppressoris both cysteine and methionine.

Furthermore, the invention provides a method for titrating theexpression of a fungal cell essential polynucleotide product, saidmethod including the steps of:

(a) inactivating a first copy of a polynucleotide in diploid fungalcells by recombination using a PCR-based polynucleotide disruptioncassette, thereby providing heterozygous diploid fungal cells;

(b) modifying the second copy of the polynucleotide in the heterozygousdiploid fungal cells by recombination using a PCR-based promoterswapping cassette including a nucleotide sequence encoding a MET3promoter, such that expression of the second copy of the polynucleotideis regulated by said promoter;

(c) adding varying concentrations of a promoter suppressor whichmodulates said MET3 promoter; and

(d) correlating cell growth with each of said concentrations, therebyascertaining the amount of promoter suppressor required to result in aparticular reduction in cell growth.

Furthermore, the invention provides a method for titrating theexpression of a fungal cell essential polynucleotide product, saidmethod including the steps of:

(a) inactivating a first copy of a polynucleotide in diploid fungalcells by recombination using a PCR-based polynucleotide disruptioncassette, thereby providing heterozygous diploid fungal cells;

(b) modifying the second copy of the polynucleotide in the heterozygousdiploid fungal cells by recombination using a PCR-based promoterswapping cassette including a nucleotide sequence encoding a MET3promoter, such that expression of the second copy of the polynucleotideis regulated by said promoter;

(c) adding varying concentrations of a promoter suppressor whichmodulates said MET3 promoter; and

(d) correlating cell growth with each of said concentrations, therebyascertaining the amount of promoter suppressor required to result in aparticular reduction in cell growth,

wherein the promoter suppressor is methionine, wherein the promotersuppressor is cysteine, and/or wherein the promoter suppressor is bothcysteine and methionine.

Also provided herein is a nucleic acid molecule microarray having aplurality of nucleic acid molecules, said plurality including at leastone nucleic acid molecule having a nucleotide sequence that ishybridizable under stringent conditions to a target nucleotide sequenceselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11.

The invention also provides a fusion protein having a fragment of afirst polypeptide fused to a second polypeptide, said fragmentconsisting of at least 5 consecutive residues of an amino acid sequenceselected from one of SEQ ID NO: 12 to SEQ ID NO:22.

Additionally provided herein is a method of producing a polypeptide,said method comprising introducing into a cell, a vector comprising apromoter operably linked to a nucleotide sequence encoding a polypeptideconsisting of an amino acid sequence selected from the group consistingof one of SEQ ID NO:12 to 22; and culturing the cell such that thenucleotide sequence is expressed.

Additionally, the invention provides a method for identifying a compoundwhich modulates the activity of a polynucleotide product encoded by anucleic acid molecule including a nucleotide sequence selected from thegroup consisting of one of SEQ ID NO: 1 to 11; said method including:

(a) contacting said polynucleotide product with a compound; and

(b) determining whether said compound modulates the activity of saidpolynucleotide product.

Also included herein is a method for identifying a compound whichmodulates the activity of an essential polynucleotide product saidmethod including the steps of:

(a) inactivating a first copy of a polynucleotide in diploid fungalcells by recombination using a PCR-based polynucleotide disruptioncassette, thereby providing heterozygous diploid fungal cells;

(b) modifying the second copy of the polynucleotide in the heterozygousdiploid fungal cells by recombination using a PCR-based promoterswapping cassette including a nucleotide sequence encoding a MET3promoter, such that expression of the second copy of the polynucleotideis regulated by said promoter

(c) contacting said cells with a compound; and

(d) determining whether said compound modulates the activity of saidpolynucleotide product.

Additionally provided is a method of eliciting an immune response in ananimal including introducing into the animal a composition including anisolated polypeptide, the amino acid sequence of which includes at least6 consecutive residues of one of SEQ ID NO: 12 to 22 or one of SEQ IDNO: 48 to SEQ ID NO: 73.

Also included is a method of identifying a compound or binding partnerthat binds to a polypeptide having an amino acid sequence selected fromthe group consisting of one of SEQ ID NO: 12 to 22 or one of SEQ ID NO48 to SEQ ID NO 73 or a fragment thereof, said method including:

(a) contacting the polypeptide or fragment thereof with a plurality ofcompounds or a preparation comprising one or more binding partners; and

(b) identifying a compound or binding partner that binds to thepolypeptide or fragment thereof.

Furthermore, provided herein is a method for identifying a compoundhaving the ability to inhibit growth of Candida albicans, said methodincluding the steps of:

(a) reducing the level or activity of a polynucleotide product encodedby a nucleic acid selected from group consisting of SEQ ID NO: 1 to 11in Candida albicans cells relative to a wild type cells, wherein saidreduced level is not lethal to said cells;

(b) contacting said cell with a compound; and

(c) determining whether said compound inhibits the growth of said cells.

Furthermore, provided herein is a method for inhibiting growth ofCandida albicans cells comprising contacting the cells with a compoundthat (i) reduces the level of or inhibits the activity of a nucleotidesequence selected from the group consisting of SEQ ID NO 1 to 11 or (ii)reduces the level of or inhibits the activity of a polynucleotideproduct encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1 to 11, alternatively wherein said compound isan antisense molecule.

Additionally included herein is a method for treating an infection of asubject by Candida albicans comprising administering a pharmaceuticalcomposition including a therapeutically effective amount of a compoundthat reduces the activity or level of a polynucleotide product encodedby a nucleic acid including a sequence selected from the groupconsisting of SEQ ID NO: 1 to 11 and a pharmaceutically acceptablecarrier to said subject.

Also provided is a pharmaceutical composition including atherapeutically effective amount of an agent which reduces the activityor level of a polynucleotide product encoded by a nucleic acid selectedfrom the group consisting of SEQ ID NO 1 to 11 in a pharmaceuticallyacceptable carrier.

The present invention also provides structure coordinates of thehomology model of the CaYLR100w polypeptide (SEQ ID NO:12) provided inFIG. 27. The complete coordinates are listed in Table 8. The model ofthe present invention further provide a basis for designing stimulatorsand inhibitors or antagonists of one or more of the biological functionsof CaYLR100w, or of mutants with altered specificity.

The present invention also provides structure coordinates of thehomology model of the CaYDR341c polypeptide (SEQ ID NO:13) provided inFIG. 30. The complete coordinates are listed in Table 9. The modelspresent in this invention further provide a basis for designingstimulators and inhibitors or antagonists of one or more of thebiological functions of CaYDR341c, or of mutants with alteredspecificity.

The present invention also provides structure coordinates of thehomology model of the CaYOL010w polypeptide (SEQ ID NO:19) provided inFIG. 33. The complete coordinates are listed in Table 10. The modelspresent in this invention further provide a basis for designingstimulators and inhibitors or antagonists of one or more of thebiological functions of CaYOL010w, or of mutants with alteredspecificity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: alignment of C. albicans essential polypeptides with S.cervisiae and other sequences. FIG. 1A provides an alignment between theencoded CaYLR100w polypeptide (“A”; SEQ ID NO:12) with P1_(—)170752(“B”; SEQ ID NO:23) and AAB19089 (“C”; SEQ ID NO:24); FIG. 1B providesan alignment between the encoded CaYDR341c polypeptide (“A”; SEQ IDNO:13) with P1_(—)168653 (“B”; SEQ ID NO:25) and AAB94675 (“C”; SEQ IDNO:26); FIG. 1C provides an alignment between the encoded CaYLR022cpolypeptide (“A”; SEQ ID NO: 14) with P1_(—)178714 (“B”; SEQ ID NO:27)and AAB42957 (“C”; SEQ ID NO:28); FIG. 1D provides an alianment betweenthe encoded CaYOL077c polypeptide (“A”; SEQ ID NO:15) with P1_(—)182338(“B”; SEQ ID NO:29) and AAB62453 (“C”; SEQ ID NO:30); FIG. 1E providesan alianment between the encoded CaYNL132w polypeptide (“A”; SEQ IDNO:16) with P1_(—)137216 (“B”; SEQ ID NO:31) and AAB93917 (“C”; SEQ IDNO:32); FIG. 1F provides an alianment between the encoded CaYGR145wpolypeptide (“A”; SEQ ID NO:17) with P1_(—)150732 (“B”; SEQ ID NO:33)and AAB95680 (“C”; SEQ ID NO:26); FIG. 1G provides an alignment betweenthe encoded CaYDR412w polypeptide (“A”; SEQ ID NO:18) with P1_(—)142340(“B”; SEQ ID NO:35) and AAW33110 (“C”; SEQ ID NO:36); FIG. 1H providesan alianment between the encoded CaYOL010w polypeptide (“A”; SEQ IDNO:19) with P1_(—)182291 (“B”; SEQ ID NO:37) and AAW60075 (“C”; SEQ IDNQ:38); FIG. 1I provides an alianment between the encoded CaYOR004wpolypeptide (“A”; SEQ ID NO:20) with P1_(—)161797 (“B”; SEQ ID NO:39)and AAG48012 (“C”; SEQ ID NO:40); FIG. 1J provides an alianment betweenthe encoded CaYOR056c polypeptide (“A”; SEQ ID NO:21) with P1_(—)182387(“B”; SEQ ID NO:41) and AAB09929 (“C”; SEQ ID NO:42); FIG. 1K providesan alianment between the encoded CaYRL009W polypeptide (“A”; SEQ IDNO:22) with P1_(—)178703 (“B”; SEQ ID NO:43) and AAB43803 (“C”; SEQ IDNO:44); and FIG. 1L provides an alignment between the encoded CaYJR072cpolypeptide (“A”; SEQ ID NO:45) with P1_(—)149006 (“B”; SEQ ID NQ:46)and AAG46965 (“C”; SEQ ID NQ:47).

FIG. 2: alignment of C. albicans essential polypeptides with A.fumigatus sequences. FIG. 2A provides an alignment between the encodedCaYLR100w polypeptide (“A”; SEQ ID NO:12) with two versions of theanalogous sequence in A. fumigatus (“B”and “C”; SEQ ID NO:48 and 49);FIG. 2B provides an alinnment between the encoded CaYDR341c polypeptide(“A”; SEQ ID NO:13) with three versions of the analogous sequence in A.fumigatus (“B”, “C”, and “D”; SEQ ID NO:50, 51 and 52); FIG. 2C providesan alianment between the encoded CaYLR022c polypeptide (“A”; SEQ IDNO:14) with two versions of the analogous sequence in A. fumigatus(“B”and “C”; SEQ ID NO:53 and 54); FIG. 2D provides an alignment betweenthe encoded CaYOL077c polypeptide (“A”; SEQ ID NO:15) with two versionsof the analogous sequence in A. fumigatus (“B”and “C”; SEQ ID NO:55 and56); FIG. 2E provides an alignment between the encoded CaYNL132wpolypeptide (“A”; SEQ ID NO:16) with two versions of the analogoussequence in A. fumigatus (“B”and “C”; SEQ ID NO:57 and 58); FIG. 2Fprovides an alignment between the encoded CaYDR412w polypeptide (“A”;SEQ ID NO: 18) with two versions of the analogous sequence in A.fumigatus (“B”and “C”; SEQ ID NO:62 and 63); FIG. 2G provides analignment between the encoded CaYOR004w polypeptide (“A”; SEQ ID NO:20)with two versions of the analogous sequence in A. fumigatus (“B”and “C”;SEQ ID NO:64 and 65); FIG. 2H provides an alignment between the encodedCaYOR056c polypeptide (“A”; SEQ ID NO:21) with three versions of theanalogous sequence in A. fumigatus (“B”, “C”, and “D”; SEQ ID NO:66, 67,and 68); FIG. 2I provides an alignment between the encoded CaYLR009wpolypeptide (“A”; SEQ ID NO:22) with two versions of the analogoussequence in A. fumigatus (“B”and “C”; SEQ ID NO:69 and 70); FIG. 2Jprovides an alignment between the encoded CaYOL010w polypeptide (“A”;SEQ ID NO: 19) with three versions of the analogous sequence in A.fumigatus (“B”, “C”, and “D”; SEQ ID NO:71, 72, and 73); FIG. 2Kprovides an alignment between the encoded CaYOL010w polypeptide (“A”;SEQ ID NO:19) two versions of the analogous sequence in A. fumigatus((“B”and “C”; SEQ ID NO:71 and 73); and FIG. 2L provides an alignmentbetween the encoded CaYJR072c polypeptide (“A”; SEQ ID NO:45) with sixversions of the analogous sequence in A. fumigatus (“B”, “C”, “D”, “E”,“F”, and “G”; SEQ ID NO:74, 75, 76, 77, 78, and 79).

FIG. 3: PCR-based polynucleotide disruption in C. albicans.

FIG. 4: PCT confirmation of AAH/disruption strains that were obtainedvia PRC-based polynucleotide disruption in C. albicans.

FIG. 5: Plasmid maps of pUMP and pAMP used for promoter swapping.

FIG. 6: Scheme of promoter swapping.

FIG. 7: Confirmation PCR for C. albicans MET3P-E RG1/erg1:: ARG4strains.

FIG. 8: Down regulation phenotypes by methionine and cysteine.

FIG. 9: Methionine titration of MET3P-ERG1 construct.

FIG. 10: Sensitivity of MET3P-ERG1 cells to terbinafine in the absenceand presence of methionine.

FIG. 11: The polynucleotide sequence (SEQ ID NO:1) and deduced aminoacid sequence (SEQ ID NO:12) of the novel fungal essential gene,CaYLR100w (also referred to as FCG5), of the present invention. TheCaYLR100w polypeptide (SEQ ID NO:12) is encoded by nucleotides 1 to 1038of SEQ ID NO:1 and has a predicted molecular weight of 39.0 kDa. Theconserved 3-keto sterol reductase catalytic residues, Y247 and S183, aredenoted by light shading.

FIG. 12: The polynucleotide sequence (SEQ ID NO:2) and deduced aminoacid sequence (SEQ ID NO:13) of the novel fungal essential gene,CaYDR341c (also referred to as FCG6), of the present invention. TheCaYDR341c polypeptide (SEQ ID NO:13) is encoded by nucleotides 1 to 1866of SEQ ID NO:2 and has a predicted molecular weight of 70.8 kDa. Theconserved amino acids comprising the adenylate binding site ofarginyl-tRNA synthetases, P151-H161, is denoted by light shading. Theconserved amino acids comprising the Ω loop of arginyl-tRNA synthetases,S496-G502, is denoted by double underlining.

FIG. 13: The polynucleotide sequence (SEQ ID NO:3) and deduced aminoacid sequence (SEQ ID NO:14) of the novel fungal essential gene,CaYLR022c (also referred to as FCG7), of the present invention. TheCaYLR022c polypeptide (SEQ ID NO:14) is encoded by nucleotides 1 to 765of SEQ ID NO:3 and has a predicted molecular weight of 29.2 kDa.

FIG. 14: The polynucleotide sequence (SEQ ID NO:4) and deduced aminoacid sequence (SEQ ID NO:15) of the novel fungal essential gene,CaYOL077c (also referred to as FCG8), of the present invention. TheCaYOL077c polypeptide (SEQ ID NO:15) is encoded by nucleotides 1 to 876of SEQ ID NO:4 and has a predicted molecular weight of 34.0 kDa.

FIG. 15: The polynucleotide sequence (SEQ ID NO:5) and deduced aminoacid sequence (SEQ ID NO:16) of the novel fungal essential gene,CaYNL132w (also referred to as FCG10), of the present invention. TheCaYNL132w polypeptide (SEQ ID NO:16) is encoded by nucleotides 1 to 3126of SEQ ID NO:5 and has a predicted molecular weight of 117.3 kDa.

FIG. 16: The polynucleotide sequence (SEQ ID NO:6) and deduced aminoacid sequence (SEQ ID NO:17) of the novel fungal essential gene,CaYGR145w (also referred to as FCG12), of the present invention. TheCaYGR145w polypeptide (SEQ ID NO:17) is encoded by nucleotides 1 to 2250of SEQ ID NO:6 and has a predicted molecular weight of 85.0 kDa.

FIG. 17: The polynucleotide sequence (SEQ ID NO:7) and deduced aminoacid sequence (SEQ ID NO:18) of the novel fungal essential gene,CaYDR412w (also referred to as FCG13), of the present invention. TheCaYDR412w polypeptide (SEQ ID NO:18) is encoded by nucleotides 1 to 804of SEQ ID NO:7 and has a predicted molecular weight of 31.3 kDa.

FIG. 18: The polynucleotide sequence (SEQ ID NO:8) and deduced aminoacid sequence (SEQ ID NO:19) of the novel fungal essential gene,CaYOL010w (also referred to as FCG14), of the present invention. TheCaYOL010w polypeptide (SEQ ID NO:19) is encoded by nucleotides 1 to 1113of SEQ ID NO:8 and has a predicted molecular weight of 40.6 kDa. Theconserved RNA 3′-terminal phosphate cyclase comprising the nucleotidebinding site residues, R158-V168, are denoted by light shading.

FIG. 19: The polynucleotide sequence (SEQ ID NO:9) and deduced aminoacid sequence (SEQ ID NO:20) of the novel fungal essential gene,CaYOR004w (also referred to as FCG15), of the present invention. TheCaYOR004w polypeptide (SEQ ID NO:20) is encoded by nucleotides 1 to 771of SEQ ID NO:9 and has a predicted molecular weight of 29.5 kDa.

FIG. 20: The polynucleotide sequence (SEQ ID NO:10) and deduced aminoacid sequence (SEQ ID NO:21) of the novel fungal essential gene,CaYOR056c (also referred to as FCG16), of the present invention. TheCaYOR056c polypeptide (SEQ ID NO:21) is encoded by nucleotides 1 to 1398of SEQ ID NO:10 and has a predicted molecular weight of 52.6 kDa.

FIG. 21: The polynucleotide sequence (SEQ ID NO:11) and deduced aminoacid sequence (SEQ ID NO:22) of the novel fungal essential gene,CaYLR009w (also referred to as FCG17), of the present invention. TheCaYLR009w polypeptide (SEQ ID NO:22) is encoded by nucleotides 1 to 585of SEQ ID NO:11 and has a predicted molecular weight of 23.1 kDa.

FIG. 22. TLC autoradiogram of labeled sterols from strainscaerglΔ/P_(MET3-)CaERG1 (A) and fcg5Δ/P_(MET3-)FCG5 (B). The position ofergosterol, lanosterol and squalene are indicated by arrows, aslabelled. The squalene epoxidase (Erg1) inhibitor, terbinafine (“Ter”),was added to both a set of control tubes and a set of Met/Cys-containingtubes for a 90 minute induction period. Note that [14C]-cholesterol wasused as a migration standard for ergosterol in Lane 11. The resultsdemonstrate that CaYLR100w is a 3-keto sterol reductase. Additionalexperimental details may be found in Example 11, and described elsewhereherein.

FIG. 23. Growth curve of strains caerglΔ/P_(MET3-)CaERG1 (A) andfcg5Δ/P_(MET3-)FCG5 (B) in the absence and presence of methionine andcysteine. The results further demonstrate that CaYLR100w is a 3-ketosterol reductase. Additional experimental details may be found inExample 11, and described elsewhere herein.

FIG. 24. Incorporation of [¹⁴C]-acetate into ergosterol and lanosterolin strains caerglΔ/P_(MET3-)CaERG1 (A) and fcg5Δ/_(PMET3-)FCG5 (B) inthe absence and presence of methionine and cysteine. The results furtherdemonstrate that CaYLR100w is a 3-keto sterol reductase involved in C-4sterol demethylation. Additional experimental details may be found inExample 11, and described elsewhere herein.

FIG. 25. Incorporation of radio-labeled arginine and leucine intoproteins in cells of fcg6Δ/_(PMET3-)FCG6 in the absence and presence ofmethionine and cysteine. (A.) raw counts (cpm) of [3H]-leu and [3H]-argin the absence and presence of methionine and cysteine. (B.) percentprotein synthesis by using the counts of untreated cells as 100%. Theresults demonstrate that CaYDR341c is involved in whole cell proteinsynthesis. Additional experimental details may be found in Example 12,and described elsewhere herein.

FIG. 26. Sequence alignment of the conceptual translated sequence ofCaYLR100w (FCG5) polypeptide of the present invention (SEQ ID NO:12)with porcine carbonyl reductase (Protein Data Bank entry 1HU4; GenbankAccession No. gil5826210; SEQ ID NO:251). The alignment was used as thebasis for building the CaYLR100w homology model described herein. Thecoordinates of the CaYLR100w model are provided in Table 8. Amino acidsconserved from the short chain dehydrogenase/reductase (SDR) catalytictriad are highlighted with an asterisk (*). Homologous residues in thefunctional active site domain that are conserved by identity areillustrated in bold.

FIG. 27 shows the three-dimensional homology model of the CaYLR100w(FCG5) polypeptide of the present invention (SEQ ID NO:12). The model isbased upon an alignment to a structural homologue porcine carbonylreductase (Protein Data Bank entry 1HU4; Genbank Accession No.gil15826210; SEQ ID NO:251) that was used as the basis for building theCaYLR100w homology model. The coordinates of the CaYLR100w model areprovided in Table 8.

FIG. 28 shows a comparison of the energy of CaYLR100w 3-keto sterolreductase homology model to the crystal structure of the porcinecarbonyl reductase (Protein Data Bank entry 1HU4; Genbank Accession No.gil15826210; SEQ ID NO:251) on which the CaYLR100w model was based. TheCaYLR100w homology model is represented by the dotted (dashed) line andthe porcince carbonyl reductase crystal structure is represented by thesolid line.

FIG. 29: Sequence alignment of the conceptual translated sequence ofCaYDR341c (FCG6) polypeptide of the present invention (SEQ ID NO:13)with Saccharomyces cerevisiae arginyl-tRNA synthetase, (chain A)(Protein Data Bank entry 1F7U; Genbank Accession No. gil14719542; SEQ IDNO:252). The alignment was used as the basis for building the CaYDR341chomology model described herein. The coordinates of the CaYDR341c modelare provided in Table 9. Amino acids defining the ADP binding siteregion and Ω loop in both the model and the 1F7U structure arehighlighted with either asterisk (“*”), or plus (“+”) sign,respectively. Homologous residues in the functional active site domainthat are conserved by identity are illustrated in bold.

FIG. 30 shows the three-dimensional homology model of the CaYDR341c(FCG6) polypeptide of the present invention (SEQ ID NO:13). The model isbased upon an alignment to a structural homologue Saccharomycescerevisiae arginyl-tRNA synthetase, (chain A) (Protein Data Bank entry1F7U; Genbank Accession No. gil14719542; SEQ ID NO:252) that was used asthe basis for building the CaYDR341c homology model. The coordinates ofthe CaYDR341c model are provided in Table 9.

FIG. 31 shows a comparison of the energy of the CaYDR341c arginyl-tRNAsynthetase homology model to the crystal structure of the Saccharomycescerevisiae arginyl-tRNA synthetase, (chain A) (Protein Data Bank entry1F7U; Genbank Accession No. gil14719542; SEQ ID NO:252) on which theCaYDR341c model was based. The CaYDR341c homology model is representedby the dotted (dashed) line and the Saccharomyces cerevisiaearginyl-tRNA synthetase crystal structure is represented by the solidline.

FIG. 32: Sequence alignment of the conceptual translated sequence ofCaYOL010w (FCG14) of the present invention (SEQ ID NO:19) withEscherichia coli RNA 3′-terminal phosphate cyclase (Protein Data Bankentry 1QMH; Genbank Accession No. gil12644279; SEQ ID NO:253). Thealignment was used as the basis for building the CaYOL010w homologymodel described herein. The coordinates of the CaYOL010w model areprovided in Table 10. Amino acids defining the nucleotide binding siteregion in both the model and the 1 QMH structure are highlighted with anasterisk (“*”). Homologous residues in the functional active site domainthat are conserved by identity are illustrated in bold.

FIG. 33 shows the three-dimensional homology model of the CaYOL010w(FCG6) polypeptide of the present invention (SEQ ID NO:19). The model isbased upon an alignment to a structural homologue Escherichia coli RNA3′-terminal phosphate cyclase (Protein Data Bank entry 1QMH; GenbankAccession No. gi|12644279; SEQ ID NO:253) that was used as the basis forbuilding the CaYOL010w homology model. The coordinates of the CaYOL010wmodel are provided in Table 10.

FIG. 34 shows a comparison of the energy of the CaYOL010w arginyl-tRNAsynthetase homology model to the crystal structure of the Escherichiacoli RNA 3′-terminal phosphate cyclase (Protein Data Bank entry 1 QMH;Genbank Accession No. gi|12644279; SEQ ID NO:253) on which the CaYOL010wmodel was based. The CaYOL010w homology model is represented by thedotted (dashed) line and the Saccharomyces cerevisiae arginyl-tRNAsynthetase crystal structure is represented by the solid line.

Table 1 provides a summary of the percent similarity between the Candidaalbicans CURFs of the present invention to the homologous sequences inS. cerevisiae.

Table 2 provides a summary of the percent identity to sequenceshomologous to te fungal CURFs of the present invention in addition tothe functional annotation of the same.

Table 3 identifies the Genbank Accession No. and/or patent or patentapplication number of homologous sequences that aligning with the CURFsof the present invention.

Table 4 lists the SEQ ID Nos. for the PCR primers used for knockoutexperiments.

Table 5 lists the predicted function of the novel conserved essentialfungal polypeptides of the present invention.

Table 6 lists the SEQ ID NOs of the MET3 promoter swapping primers whichmay be used to remove the promoters associated with the essentialpolynucleotides encoded by SEQ ID NO: 1 through to SEQ ID NO: 11.

Table 7 shows the results of experiments designed to assess thesensitivity of the MET3P-ERG1 construct to antifungal drugs.

Table 8 provides the structural coordinates of the three dimensionalstructure of the CaYLR100w (FCG5) polypeptide of the present invention(SEQ ID NO:12).

Table 9 provides the structural coordinates of the three dimensionalstructure of the CaYDR341c (FCG6) polypeptide of the present invention(SEQ ID NO:13).

Table 10 provides the structural coordinates of the three dimensionalstructure of the CaYOL010w (FCG14) polypeptide of the present invention(SEQ ID NO:19).

Table 11: Illustrates the preferred hybridization conditions for thepolynucleotides of the present invention. Other hybridization conditionsmay be known in the art or are described elsewhere herein.

Table 12: Provides a summary of various conservative substitutionsencompassed by the present invention.

DETAILED DESCRIPTION OF THE INVENTION Nucleic Acids Encoding EssentialPolynucleotides

The present invention is directed to polynucleotides that encodepolypeptides that have been shown to be essential for Candida albicansgrowth and survival. Such polypeptides are referred to herein as FungalConserved Genes (“FCG”), or Conserved Unknown Reading Frames (“CURFs”).

The present invention is also directed to the homologous polynucleotideand polypeptide sequences of the fungal conserved genes of the presentinvention in A. fumigatus.

In one embodiment, the invention provides nucleotide sequences encodingpolypeptides that are essential to Candida albicans. A polynucleotide isgenerally considered essential when the viability of the organism issubstantially coupled to or dependent on the expression of thepolynucleotide. Such nucleotide sequences include, but are not limitedto, SEQ ID NO: 1 through SEQ ID NO:11, fragments and homologues thereof.

The term “essential polynucleotides” refers to a nucleotide sequencethat encodes a polynucleotide product (mRNA or protein) having afunction which is required for cell viability. The term “essentialprotein” refers to a polypeptide that is encoded by an essentialpolynucleotide and has a function that is required for cell viability.Accordingly, a mutation that disrupts the function of the essentialpolynucleotide or essential proteins results in a loss of viability ofcells harboring the mutation.

In another embodiment, the invention provides an isolated nucleic acidmolecule having a nucleotide sequence encoding a polypeptide essentialto Candida albicans, wherein said polypeptide comprises an amino acidsequence of one of SEQ ID NO: 12 to 22.

The invention also includes the complements of SEQ ID 1 to SEQ ID 11 andfragments thereof.

A nucleic acid molecule is said to be the “complement” of anothernucleic acid molecule if two single-stranded polynucleotides anneal bybases-pairing. For example, 5′-ACG-3′ pairs with its complement3′-TGC-5′. 100% complementarity occurs when every nucleotide of one ofthe molecules is complementary to a corresponding nucleotide of theother.

The essential polynucleotides represent potential drug targets forCandida albicans and can be used individually or as a collection invarious methods of drug screening herein described. The essentialpolynucleotides provided were found to be homologous withpolynucleotides in S. cervisiae and/or other fungi and lackedsignificant similarity with human polynucleotides. Such an essentialpolynucleotide set can be conveniently investigated as a group in a drugscreening program.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising a nucleotide sequence encoding a polypeptideor a biologically active ribonucleic acid (RNA). The term can furtherinclude nucleic acid molecules comprising upstream, downstream and/orintron nucleotide sequences. The term “open reading frame (ORF)” means aseries of nucleotide triplets coding for amino acids without anytermination codons and the triplet sequence is translatable into proteinusing the codon usage information appropriate for a particular organism.

As used herein, the terms “polynucleotide”, “nucleotide sequence”,“nucleic acid molecule”, “nucleic acid” or “oligonucleotide” are usedinterchangeably and refer to a heteropolymer of nucleotides or thesequence of these nucleotides. These phrases also refer to DNA or RNA ofgenomic or synthetic origin which may be single-stranded ordouble-stranded and may represent the sense or the antisense strand. Inthe sequences herein, A is adenine, C is cytosine, G is guanine, T isthymine and N is G, A, C, or T(U). It is contemplated that where thepolynucleotide is RNA, the T (thymine) in the sequences provide hereinmay be substituted with U (uracil).

By “isolated” polynucleotide(s) is intended a polynucleotide, DNA orRNA, which has been removed from its native environment. For example,recombinant DNA molecules contained in a vector are considered isolatedfor the purposes of the present invention. Further examples of isolatedDNA molecules include recombinant DNA molecules maintained inheterologous host cells or purified (partially or substantially) DNAmolecules in solution. Isolated RNA molecules include in vivo or invitro RNA transcripts of the DNA molecules of the present invention.Isolated polynucleotide molecules according to the present inventionfurther include such molecules produced synthetically.

“Substantially purified” refers to nucleic or amino acid sequences thatare removed from their natural environment, isolated or separated, andare at least 60% free, preferably 75% free, and most preferably 90% freefrom other components with which they are naturally associated.

The invention also provides a composition including SEQ ID NO: 1 to SEQID NO: 11 or fragments or variants thereof. A “composition including agiven polynucleotide sequence or polypeptide sequence” refers broadly toany composition containing the given polynucleotide or polypeptidesequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions including polynucleotide sequences, andpolynucleotide sequences encoding essential polynucleotides may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be associated with a stabilizing agent such asa carbohydrate. In hybridizations, the probe may be deployed in anaqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS)and other components (e.g., Denhardt's solution, dry milk, salmon spermDNA, etc.).

Fragment nucleic acid molecules may encode significant portion(s) of, ormost of, the polypeptides of the present invention. A fragment maycomprise up to the entire length of the defined sequence, minus onenucleotide. For example, a fragment may comprise from 5 to 3128contiguous nucleotides. Fragments of the target polynucleotides of theinvention can also refer to portions of the coding regions of suchnucleic acid molecules that encode functional domains such as signalsequences, extracellular domains, transmembrane domains and cytoplasmicdomains.

Fragment nucleic molecules of the present invention also include primersand probes. “Probe” refers to polynucleotides encoding essentialpolynucleotides of the invention, their complements or fragmentsthereof, which are used to detect allelic or related polynucleotides.Probes are isolated oligonucleotides or polynucleotides attached to adetectable label or reporter molecule. Typical labels includeradioactive isotopes, chemiluminescent agents and enzymes (see, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratories (1989), which is incorporated herein by reference inits entirety).

Probes and primers as used in the present invention typically compriseat least 10 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 10, 20, 25, 30, 40,50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed polynucleotides. Probes and primers may be considerably longerthan these examples, and it is understood that any length supported bythe specification including the tables, FIGs and Sequence Listing may beused.

The primers of the invention may be used in conjunction with thepolymerase chain reaction (PCR). PCR exploits certain features of DNAreplication. DNA polymerase uses single-stranded DNA as a template forthe synthesis of a complementary new strand. These single-stranded DNAtemplates can be produced by heating double-stranded DNA to temperaturesnear boiling. DNA polymerase also requires a small section ofdouble-stranded DNA to initiate (“prime”) synthesis. Therefore, thestarting point for DNA synthesis can be specified by supplying a PCRprimer that anneals to the template at that point. Methods for designingPCR primers are well-known in the art. (See, for example, Innis et al.,PCR Protocols: A Guide to Methods and Applications, Academic, N.Y.(1990), herein incorporated by reference). Computer programs may also beused to design PCR primers. For example, Primer3.

In addition to the nucleotide sequences of Candida albicans describedabove, homologues of these target polynucleotide sequences in otherspecies can be identified and isolated by molecular biologicaltechniques well-known in the art and without undue experimentation.

To isolate homologous target polynucleotides, the C. albicans targetpolynucleotide sequences described above can be labeled and used toscreen a cDNA library constructed from mRNA obtained from the organismof interest. Hybridization conditions should be of a lower stringencywhen the cDNA library is derived from an organism different from thetype of organism from which the labeled sequence was derived. cDNAscreening can also identify clones derived from alternatively splicedtranscripts in the same or different species. Alternatively, the labeledfragment can be used to screen a genomic library derived from theorganism of interest, again, using appropriately stringent conditions.Reduced stringency conditions will be well-known to those of skill inthe art and will vary predictably depending on the specific organismsfrom which the library and the labeled sequences are derived. (See, forexample, Sambrook et al., supra)

Further, a homologous target polynucleotide sequence can be isolated byperforming a polymerase chain reaction (PCR) using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequences within the target polynucleotide of interest. The template forthe reaction can be cDNA obtained by reverse transcription of mRNAprepared from the organism of interest. The PCR product can be subclonedand sequenced to ensure that the amplified sequences represent thesequences of a homologous target polynucleotide sequence.

Alternatively, homologous target polynucleotides or polypeptides may beidentified by searching a dataset to identify sequences having a desiredlevel of homology to an essential polynucleotide of the invention. Avariety of such databases are available to those skilled in the artincluding GenBank. In various embodiments, the databases are screened toidentify nucleic acids with at least 97%, at least 95%, at least 90%, atleast 85%, at least 80%, at least 70%, at least 60%, at least 50%, atleast 40%, or at least 30% identity to an essential polynucleotide ofthe invention.

“Homologous sequences” or “homologues” as used herein are thosesequences in which a first amino acid or nucleotide sequence contains asufficient or minimum number of identical or equivalent (e.g., an aminoacid residue which has a similar side chain) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences share commonstructural domains or motifs and/or a common functional activity whenoptimally aligned. For example, amino acid or nucleotide sequences whichshare common structural domains have at least about 30-40% homology,preferably 40-50% homology, more preferably 50-60%, and even morepreferably 60-70%, 70-80%, or 80-90% or 95% homology across the aminoacid sequences of the domains and contain at least one and preferablytwo structural domains or motifs. Furthermore, amino acid or nucleotidesequences which share at least 30-40%, preferably 40-50%, morepreferably 50-60%, 60-70%, 70-80%, or 80-90% or 95% homology and share acommon functional activity are homologous.

In one embodiment, the invention provides for homologues of theessential polynucleotides of the invention encoded by SEQ ID NO: 1through to SEQ ID NO: 11 in species including, but not limited to,Aspergillus fumigatus, Aspergillus falvus, Aspergillus niger,Coccidiodes immitis, Cryptoccoccus neoformans, Histoplasma capsulatum,Phytophthora infestans, Puccinia seconditii, Pneumocystis carinii or anyspecies falling within the genera of any of the above species. Otheryeasts in the genera of Candida, Saccharomyces, SchizosaccharomycesSporobolomyces, Torulopsis, Trichosporon, Tricophyton, Dermatophytes,Microsproum, Wickerhamia, Ashbya, Blastomyces, Citeromyces,Crebrothecium, Cryptococcus, Debaryomyces, Endomycopsis, Geotrichum,Hansenula, Kloecker, Kluveromyces, Libomyces, Pichia, Rhodosporidium,Rhodotorula, and Yarrowia are also contemplated.

Preferably, homologues of the essential polynucleotides of the inventionencoded by SEQ ID NO: 1 through to SEQ ID NO: 11 are from Absidiacorymbigera, Aspergillus flavis, Aspergillus fumigatus, Aspergillusniger, Botrytis cinerea, Candida dublinensis, Candida glabrata, Candidakrusei, Candia parapsilopsis, Candia tropicalis, Coccidioides immitis,Cryptococcus neoformans, Erysiphe graminis, Exophalia dermatiditis,Fusarium osysproum, Histoplasma capsulatum, Magnaporthe grisea, Mucorrouxii, Pneumocystis carinii, Puccinia graminis, Puccinia recodita,Rhizomucor pusillus, Puccinia striiformis, Rhizopus arrhizus, Septoriaavenae, Septoria nodorum, Septoria triticii, Tilletia controversa,Tilletia tritici, Trichospoon beigelii and Ustilago maydis. Particularlypreferred are homologues from Aspergillus fumigatus.

The invention also provides nucleotide sequences that are hybridizableunder stringent conditions to the polynucleotides of SEQ ID NO: 1through SEQ ID NO: 11 and that are of a species other than Saccharomycescerevisiae and Candida albicans.

The term “stringent conditions” or “hybridizable under stringentconditions” includes reference to conditions under which a probe willselectively hybridize to its target sequence, to a detectably greaterdegree than to other sequences (e.g., at least two-fold overbackground). Stringent conditions are sequence-dependent and will bedifferent in different circumstances. By controlling the stringency ofthe hybridization and/or washing conditions, target sequences can beidentified which are 100% complementary to the probe. Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected. Generally, aprobe is less than about 1000 nucleotides in length, optionally lessthan 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na+ ion, typically about 0.01 to1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984):T_(m)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. T_(m) is reduced by about 1° C. for each 1% ofmismatching; thus, T_(m), hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with 90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the thermal melting point (T_(m)) for the specific sequenceand its complement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution) it ispreferred to increase the SSC concentration so that a higher temperaturecan be used. An extensive guide to the hybridization of nucleic acids isfound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995), herein incorporated byreference. The duration of hybridization is generally less than about 24hours, usually about 4 to about 12 hours.

Additional examples of stringency conditions are shown in Table 11below: highly stringent conditions are those that are at least asstringent as, for example, conditions A-F; stringent conditions are atleast as stringent as, for example, conditions G-L; and reducedstringency conditions are at least as stringent as, for example,conditions M-R.

TABLE 11 Stin- gen- cy Con- Poly- Hyridization Wash di- nucleotideHybrid Length Temperature Temperature tion Hybrid± (bp)‡ and Buffer† andBuffer† A DNA:DNA > or equal to 50 65° C.; 1xSSC - 65° C.; or- 42° C.;0.3xSSC 1xSSC, 50% formamide B DNA:DNA <50 Tb*; 1xSSC Tb*; 1xSSC CDNA:RNA > or equal to 50 67° C.; 1xSSC - 67° C.; or- 45° C.; 0.3xSSC1xSSC, 50% formamide D DNA:RNA <50 Td*; 1xSSC Td*; 1xSSC E RNA:RNA > orequal to 50 70° C.; 1xSSC - 70° C.; or- 50° C.; 0.3xSSC 1xSSC, 50%formamide F RNA:RNA <50 Tf*; 1xSSC Tf*; 1xSSC G DNA:DNA > or equal to 5065° C.; 4xSSC - 65° C; or- 45° C.; 1xSSC 4xSSC, 50% formamide H DNA:DNA<50 Th*; 4xSSC Th*; 4xSSC I DNA:RNA > or equal to 50 67° C.; 4xSSC - 67°C.; or- 45° C; 1xSSC 4xSSC, 50% formamide J DNA:RNA <50 Tj*; 4xSSC Tj*;4xSSC K RNA:RNA > or equal to 50 70° C.; 4xSSC - 67° C.; or- 40° C.;1xSSC 6xSSC, 50% formamide L RNA:RNA <50 Tl*; 2xSSC Tl*; 2xSSC MDNA:DNA > or equal to 50 50° C.; 4xSSC - 50° C.; or- 40° C 6xSSC, 2xSSC50% formamide N DNA:DNA <50 Tn*;6xSSC Tn*;6xSSC O DNA:RNA > or equal to50 55° C.; 4xSSC - 55° C.; or- 42° C.; 2xSSC 6xSSC, 50% formamide PDNA:RNA <50 Tp*; 6xSSC Tp*; 6xSSC Q RNA:RNA > or equal to 50 60° C.;4xSSC - 60° C; 2Xssc or- 45° C.; 6xSSC, 50% formamide R RNA:RNA <50 Tr*;4xSSC Tr*; 4xSSC ‡The “hybrid length” is the anticipated length for thehybridized region(s) of the hybridizing polynucleotides. Whenhybridizing a polynucletotide of unknown sequence, the hybrid is assumedto be that of the hybridizing polynucleotide of the present invention.When polynucleotides of known sequence are hybridized, the hybrid lengthcan be determined by aligning the sequences of the polynucleotides andidentifying the region or regions of optimal sequence complementarity.Methods of aligning two or more polynucleotide sequences and/ordetermining the percent identity between two polynucleotide sequencesare well known in the art (e.g., MegAlign program of the DNA*Star suiteof programs, etc). †SSPE (1xSSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15 M NaCl anmd15 mM sodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes after hybridization is complete. Thehydridizations and washes may additionally include 5X Denhardt'sreagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA,0.5% sodium pyrophosphate, and up to 50% formamide. *Tb-Tr: Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature Tm of the hybrids there Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.) = 2(# of A + T bases) ± 4(# of G + C bases). For hybrids between 18and 49 base pairs in length, Tm(° C.) = 81.5 + 16.6(log₁₀[Na+]) + 0.41(%G + C) − (600/N), where N is the number of bases in the hybrid, and[Na+] is the concentration of sodium ions in the hybridization buffer([NA+] for 1xSSC = .165 M). ±The present invention encompasses thesubstitution of any one, or more DNA or RNA hybrid partners with eithera PNA, or a modified polynucleotide. Such modified polynucleotides areknown in the art and are more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook, J., E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel etal., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, whichare hereby incorporated by reference herein.

In another embodiment, the present invention encompasses isolatednucleic acids comprising a nucleotide sequence that has at least 40%,45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more nucleotidesequence identity to the nucleotide sequences set forth in SEQ ID NO 1to SEQ ID NO 11. The nucleotide sequences of the invention also includenucleotide sequences that encode polypeptides having at least 25%, 30%,40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or higheramino acid sequence identity or similarity to the amino acid sequencesset forth in SEQ ID NO 12 to SEQ ID NO 22.

The following terms are used to describe the sequence relationshipsbetween a polynucleotide or polypeptide of the present invention with areference polynucleotide or a polypeptide to determine sequenceidentity: (a) “reference sequence”, (b) “comparison window”, (c)“sequence identity”, and (d) “percentage of sequence identity”.

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison with a polynucleotide or a polypeptide ofthe present invention. A reference sequence may be a subset or theentirety of a specified sequence; for example, as a segment of afull-length cDNA or polynucleotide sequence, or the complete cDNA orpolynucleotide sequence.

As used herein. “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to thenucleotides or residues in the two sequences which are the same whenaligned for maximum correspondence over a specified comparison window.As used herein, “comparison window” includes reference to a contiguousand specified segment of a polynucleotide or polypeptide sequence,wherein the polynucleotide or polypeptide sequence may be compared to areference sequence and wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides/amino acids residues in length, and optionally can be 30,40, 50, 100, or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide or polypeptide sequence, a gap penalty istypically introduced and is subtracted from the number of matches.

When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988), e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif., USA).

Tolerated conservative amino acid substitutions of the present inventioninvolve replacement of the aliphatic or hydrophobic amino acids Ala,Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gln, replacement of the basic residues Lys, Arg, andHis; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservativesubstitutions provided in Table 12 below.

TABLE 12 For Amino Acid Code Replace with any of: Alanine A D-Ala, Gly,beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg,D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp,D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic D D-Asp, D-Asn, Asn, Glu, D-Glu,Gln, D-Gln Acid Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-ThrGlutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic E D-Glu,D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Acid Glycine G Ala, D-Ala, Pro,D-Pro, β-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-MetLeucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine MD-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine FD-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro,L-1-thioazolidine-4-carboxylic acid, D- or L-1-oxazolidine-4-carboxylicacid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O),L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met,Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions mayalso increase protein or peptide stability. The invention encompassesamino acid substitutions that contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the protein orpeptide sequence. Also included are substitutions that include aminoacid residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,β or γ amino acids.

Both identity and similarity can be readily calculated by reference tothe following publications: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Informatics Computer Analysis of Sequence Data, Part 1,Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution ofamino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple polynucleotides with related function and assessingthe relative penalty of each substitution to proper polynucleotidefunction. Such probabilities are often described in a matrix and areused by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) incalculating percent similarity wherein similarity refers to the degreeby which one amino acid may substitute for another amino acid withoutlose of function. An example of such a matrix is the PAM250 or BLOSUM62matrix.

Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch,J. Mol. Biol. 48: 443 (1970); by the search for similarity method ofPearson and Lipman, Proc. Nad. Acad. Sci. 85: 2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5.151.153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24:307-331 (1994). These references are herein incorporated.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the Smith-Waterman algorithm(supra) on a DeCypher system using default parameters (Matrix=Blosum62,Gap Opening penalty: 12, Gap Extension Penalty: 2).

Additionally, whether any particular nucleic acid molecule orpolypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,99.8%, or 99.9% identical to a nucleotide sequence of the presentinvention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the CLUSTALW computer program (Thompson, J. D., etal., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based onthe algorithm of Higgins, D. G., et al., Computer Applications in theBiosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment thequery and subject sequences are both DNA sequences. An RNA sequence canbe compared by converting U's to T's. However, the CLUSTALW algorithmautomatically converts U's to T's when comparing RNA sequences to DNAsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent,Window Size=5 or the length of the subject nucleotide sequence,whichever is shorter. For multiple alignments, the following CLUSTALWparameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue SpecificGaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the AlignX softwareprogram (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correctionto the percent identity results, in the instance where the subjectsequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the CLUSTALW alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino- or carboxy-terminal positions of the reference amino acidsequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at leastabout 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to,for instance, an amino acid sequence referenced in herein (SEQ IDNO:12-22) or to the amino acid sequence encoded by cDNA contained in adeposited clone, can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the CLUSTALW computer program (Thompson, J. D., etal., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based onthe algorithm of Higgins, D. G., et al., Computer Applications in theBiosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment thequery and subject sequences are both amino acid sequences. The result ofsaid global sequence alignment is in percent identity. Preferredparameters used in a CLUSTALW alignment of DNA sequences to calculatepercent identity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1,Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, GapExtension Penalty=0.1, Scoring Method=Percent, Window Size=5 or thelength of the subject nucleotide sequence, whichever is shorter. Formultiple alignments, the following CLUSTALW parameters are preferred:Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap SeparationPenalty Range=8; End Gap Separation Penalty=Off; % Identity forAlignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic ResidueGap=Off; and Transition Weighting=0. The pairwise and multple alignmentparameters provided for CLUSTALW above represent the default parametersas provided with the AlignX software program (Vector NTI suite ofprograms, version 6.0).

The present invention encompasses the application of a manual correctionto the percent identity results, in the instance where the subjectsequence is shorter than the query sequence because of N- or C-terminaldeletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polypeptide alignment. Percent identitycalculations based upon global polypeptide alignments are oftenpreferred since they reflect the percent identity between thepolypeptide molecules as a whole (i.e., including any polypeptideoverhangs, not just overlapping regions), as opposed to, only localmatching polypeptides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor N- and C-terminal truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the N-and C-termini, relative to the query sequence, the percent identity iscorrected by calculating the number of residues of the query sequencethat are N- and C-terminal of the subject sequence, which are notmatched/aligned with a corresponding subject residue, as a percent ofthe total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what may be used for the purposes of the present invention.Only residues to the N- and C-termini of the subject sequence, which arenot matched/aligned with the query sequence, are considered for thepurposes of manually adjusting the percent identity score. That is, onlyquery residue positions outside the farthest N- and C-terminal residuesof the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theCLUSTALW alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the CLUSTALWprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence, which are not matched/aligned withthe query. In this case the percent identity calculated by CLUSTALW isnot manually corrected. Once again, only residue positions outside theN- and C-terminal ends of the subject sequence, as displayed in theCLUSTALW alignment, which are not matched/aligned with the querysequence are manually corrected for. No other manual corrections arerequired for the purposes of the present invention.

In addition to the above method of aligning two or more polynucleotideor polypeptide sequences to arrive at a percent identity value for thealigned sequences, it may be desirable in some circumstances to use amodified version of the CLUSTALW algorithm which takes into accountknown structural features of the sequences to be aligned, such as forexample, the SWISS-PROT designations for each sequence. The result ofsuch a modified CLUSTALW algorithm may provide a more accurate value ofthe percent identity for two polynucleotide or polypeptide sequences.Support for such a modified version of CLUSTALW is provided within theCLUSTALW algorithm and would be readily appreciated to one of skill inthe art of bioinformatics.

Although the nucleotide sequences and amino acid sequences from S.cervisiae which are homologues of the essential polynucleotide encodedby SEQ ID NO: 1 to SEQ ID NO: 11 is mostly published, uses of suchhomologues in S. cerevisae in drug screening are not known and are thusspecifically provided by the invention. To use such nucleotide and/oramino acid sequences of S. cervisiae, public databases, such as StanfordGenomic Resources or Proteome may be used to identify and retrieve thesequences.

The nucleic acid molecules of the invention also include peptide nucleicacids (PNAs), or derivative molecules such as phosphorothioate,phosphotriester, phosphoramidate, and methylphosphonate, thatspecifically bind to single-stranded DNA or RNA in a base pair-dependentmanner (Zamecnik, P. C. et al., Proc. Natl. Acad. Sci. 75:280 284(1978); Goodchild, P. C., et al., Proc. Natl. Acad. Sci. 83:4143-4146).

PNA molecules comprise a nucleic acid oligomer to which an amino acidresidue, such as lysine, and an amino group have been added. These smallmolecules, also designated anti-polynucleotide agents, stop transcriptelongation by binding to their complementary (template) strand ofnucleic acid (Nielsen, P. E. et al., Anticancer Drug Des 8:53-63(1993)). For example, reviews of methods for synthesis of DNA, RNA andtheir analogues can be found in: Oligonucleotides and Analogues, eds. F.Eckstein, IRL Press, New York (1991); Oligonucleotide Synthesis, ed. M.J. Gait, IRL Press, Oxford, England (1984). Additionally, methods forantisense RNA technology are described in U.S. Pat. Nos. 5,194,428 and5,110,802. A skilled artisan can readily obtain these classes of nucleicacid molecules using the herein described polynucleotide sequences; see,for example, Innovative and Perspectives in Solid Phase Synthesis,Egholm, et al. pp 325-328 (1992) or U.S. Pat. No. 5,539,082.

As will be appreciated by the skilled practitioner, should the aminoacid fragment comprise an antigenic epitope, for example, biologicalfunction per se need not be maintained. The terms fungal essentialpolypeptide and fungal essential protein are used interchangeably hereinto refer to the encoded product of the fungal essential nucleic acidsequence according to the present invention.

It is another aspect of the present invention to provide modulators ofthe fungal essential polypeptides and fungal essential peptide targetswhich can affect the function or activity of fungal essentialpolynucleotides in a cell in which fungal essential polynucleotidefunction or activity is to be modulated or affected. In addition,modulators of fungal essential polypeptides can affect downstreamsystems and molecules that are regulated by, or which interact with,fungal essential polypeptides in the cell. Modulators of fungalessential polypeptides include compounds, materials, agents, drugs, andthe like, that antagonize, inhibit, reduce, block, suppress, diminish,decrease, or eliminate fungal essential polypeptides function and/oractivity. Such compounds, materials, agents, drugs and the like can becollectively termed “antagonists”. Alternatively, modulators of fungalessential polypeptides include compounds, materials, agents, drugs, andthe like, that agonize, enhance, increase, augment, or amplify fungalessential polypeptides function in a cell. Such compounds, materials,agents, drugs and the like can be collectively termed “agonists”.

As used herein the terms “modulate” or “modulates” refer to an increaseor decrease in the amount, quality or effect of a particular activity,DNA, RNA, or protein. The definition of “modulate” or “modulates” asused herein is meant to encompass agonists and/or antagonists of aparticular activity, DNA, RNA, or protein.

The present invention is also directed to polynucleotides encoding thefungal essential polynucleotides of the present invention lacking astart methionine. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. It is well known in the art that theN-terminal methionine encoded by the translation initiation codongenerally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

In specific embodiments, the present invention is directed to thefollowing polynucleotides which encode fungal essential polypeptides ofthe present invention lacking a start methionine: nucleotides 4 to 1038of SEQ ID NO:1 (CaYLR100w); nucleotides 4 to 1866 of SEQ ID NO:2(CaYDR341c); nucleotides 4 to 765 of SEQ ID NO:3 (CaYLR022c);nucleotides 4 to 876 of SEQ ID NO:4 (CaYOL077c); nucleotides 4 to 3126of SEQ ID NO:5 (CaYNL132w); nucleotides 4 to 2250 of SEQ ID NO:6(CaYGR145w); nucleotides 4 to 804 of SEQ ID NO:7 (CaYDR412w);nucleotides 4 to 1113 of SEQ ID NO:8 (CaYOL010w); nucleotides 4 to 771of SEQ ID NO:9 (CaYOR004w); nucleotides 4 to 1398 of SEQ ID NO:10(CaYOR056c); and/or nucleotides 4 to 585 of SEQ ID NO:11 (CaYLR009w).

In another embodiment, the present invention is directed torepresentative clones containing all or most of the sequence for SEQ IDNO:1 to SEQ ID NO:11 (encoding the polypeptides provided as SEQ ID NO:12to SEQ ID NO:22) that were deposited with the American Type CultureCollection (“ATCC”). The ATCC is located at 10801 University Boulevard,Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to theterms of the Budapest Treaty on the international recognition of thedeposit of microorganisms for purposes of patent procedure. Thedeposited clone is inserted in the pSport1 plasmid (Life Technologies)using the NotI and SalI restriction endonuclease cleavage sites.

Polypeptides

The polypeptides of the invention used and encompassed in the methodsand compositions of the present invention include those polypeptidesthat are encoded by the essential polynucleotide sequences as describedabove, such as the essential polynucleotide sequences set forth in SEQID NO: 1 through to SEQ ID NO: 11. The amino acid sequences of SEQ IDNO: 12 to SEQ ID NO: 22 are deduced using the codon usage of C. albicansfrom the respective nucleotide sequences of SEQ ID NO: 12 to SEQ ID NO:22. However, when expressed in an organism other than C. albicans,protein products for the target polynucleotides having the amino acidsequences of SEQ ID NO: 12 to 22 may be encoded by nucleotide sequencesthat are translated using the universal genetic code. One of skill inthe art would know the modifications that are necessary to accommodatefor such a difference in codon usage.

As used herein, the term “polypeptide” refers to any peptide or proteinincluding two or more amino acids joined to each other by peptide bondsor modified peptide bonds, i.e., peptide isosteres. “Polypeptide” refersto both short chains, commonly referred to as peptides, oligopeptides oroligomers, and to longer chains, generally referred to as proteins.“Polypeptides” may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptides” include amino acid sequences modified eitherby natural processes, such as posttranslational processing, or bychemical modification techniques which are well-known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in research literature.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.A given polypeptide may contain many types of modifications.Polypeptides may be branched, or cyclic, with or without branching.Cyclic, branched and branched-cyclic polypeptides may result fromposttranslational natural processes or may be made by synthetic methods.Modifications include but are not limited to acetylation, acylations,amidation, covalent attachment of flavin, disulfide bond formation,formation of covalent cross-links, and glycosylation. See, for instance,Proteins-structure and molecular properties, 2^(nd) Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); F. Wold,Posttranslational protein modifications: perspectives and prospects, pgs1-12 in Posttranslational covalent modification of proteins, B. C.Johnson, Ed., Academic Press, New York (1983); S. Seifter and S.Englard, Analysis for protein modifications and nonprotein cofactors,182 Methods of Enzymology 626 (1990); and S. I. Rattan et al., Proteinsynthesis, posttranslational modifications, and aging, 663 Ann NY AcadSci 48 (1992).

In addition, however, the methods and compositions of the invention alsouse and encompass proteins and polypeptides that represent functionallyequivalent polynucleotide products. Such functionally equivalentpolynucleotide products include, but are not limited to, naturalvariants of the polypeptides having an amino acid sequence set forth inSEQ ID NO; 12 to SEQ ID NO: 22.

The term “variant” (or analog) as used herein is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptide,respectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequence ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additionsand/or deletions in any combination.

Variants of the defined sequence and fragments thereof also form part ofthe present invention. Preferred variants are those that vary from thereference sequence by conservative amino acid substitutions, i.e., thosethat substitute a residue with another of like characteristics. Typicalconservative substitutions are among Ala, Val, Leu and Ile; among Serand Thr; among the acid residues Asp and Glu; among Asn and Gln; amongthe basic residues Lys and Arg; and among the aromatic residues Phe andTyr.

The term “functionally equivalent”, as utilized herein, refers to apolypeptide capable of exhibiting a substantially similar in vivoactivity as the Candida albicans essential polypeptide encoded by one ormore of the essential polynucleotide sequences described herein.Alternatively, when utilized as part of assays described herein below,the term “functionally equivalent” can refer to peptides or polypeptidesthat are capable of interacting with other cellular or extracellularmolecules in a manner substantially similar to the way in which thecorresponding portion of the target polynucleotide product wouldinteract with such other molecules. Preferably, the functionallyequivalent essential polynucleotide polypeptide of the invention arealso the same size or about the same size as a essential polynucleotidepolypeptide encoded by one or more of the essential polynucleotidesequences described herein.

Fragments of the essential polynucleotide polypeptides are also includedin the invention. A fragment is a polypeptide having an amino acidsequence that is entirely the same as part, but not all, of the aminoacid sequence of the aforementioned essential polynucleotidepolypeptides. As with essential polynucleotide polypeptides, fragmentsmay be “free-standing” or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion.

Preferred fragments of the invention are biologically active fragments.The term “active” refers to those forms of the polypeptide that retainthe biologic and/or immunologic activities of any naturally occurringpolypeptide. According to the invention, the terms “biologically active”or “biological activity” refer to a protein or peptides havingstructural, regulatory or biochemical functions of a naturally occurringmolecule. Likewise “biologically active” or “biological activity” refersto the capability of the natural, recombinant or synthetic essentialpolynucleotide peptide, or any peptide thereof, to include a specificbiological response in appropriate animals or cells and to bind withspecific antibodies.

Additionally, preferred polypeptides are those containing fragmentsincluding at least about a contiguous 5 amino acid region, morepreferably including at least a contiguous 10, 40, 50, 75 or 125 aminoacid region of a protein or fragment thereof of the present invention.In another preferred embodiment, the proteins of the present inventioninclude between about 10 and about 25 contiguous amino acid region, morepreferably between about 20 and about 50 contiguous amino acid regionand even more preferably between about 40 and about 80 contiguous aminoacid region.

Such fragments are conventionally employed by themselves or withunrelated proteins as part of fusion proteins. As used herein, a fusionprotein comprises all or part (preferably a biologically active part) ofa polypeptide of the invention operably linked to a heterologous orunrelated polypeptide. The unrelated polypeptide may be a detectablelabel for enabling detection of the polypeptide of the invention or amatrix-binding domain for immobilizing the fusion protein. The fusionproteins can be produced by standard recombinant DNA techniques.

Possible fusion protein expression vectors include but are not limitedto vectors incorporating sequences that encode beta-galactosidease andtrpE fusions, maltose-binding protein fusions (pMal series; New EnglandBiolabs), glutathionie-S-transferase fusions (pGEX series; Pharmacia)polyhistidine fusions (pET series; Novagen Inc., Madison, Wis.), andthioredoxin fusion s(pTrxFus; Invitrogen, Carlsbad, Calif.).

Expression vectors may be constructed that will express a fusion proteinincluding any protein or polypeptide of the present invention includingfragments or variants thereof. Such fusion proteins can be used, e.g.,to raise antisera against the protein, to study the biochemicalproperties of the protein, to engineer a protein exhibiting differentimmunological or functional properties, to aid in the identification orpurification of the protein, to improve the stability of arecombinantly-expressed protein or as therapeutic agents. Methods arewell-known in the art for constructing expression vectors encoding theseand other fusion proteins.

The essential polynucleotide polypeptides of the invention can beprepared in any suitable manner. The polypeptides include isolatednaturally occurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides and polypeptides produced by acombination of these methods. These methods are well understood in theart.

The present invention also provides for homologous proteins. A homologueprotein may be derived from, but not limited to, Aspergillus fumigatus,Aspergillus falvus, Aspergillus niger, Coccidiodes immitis,Cryptoccoccus neoformans, Histoplasma capsulatum, Phytophthorainfestans, Puccinia seconditii, Pneumocystis carinii or any speciesfalling within the genera of any of the above species. Other yeasts inthe genera of Candida, Saccharomyces, SchizosaccharomycesSporobolomyces, Torulopsis, Trichosporon, Tricophyton, Dernatophytes,Microsproum, Wickerhamia, Ashbya, Blastomyces, Citeromyces,Crebrothecium, Cryptococcus, Debaryomyces, Endomycopsis, Geotrichum,Hansenula, Kloecker, Kluveromyces, Libomyces, Pichia are alsocontemplated.

Preferably, homologues of the polypeptides of the invention encoded bySEQ ID NO: 1 through to SEQ ID NO: 11 are from Absidia corymbigera,Aspergillus flavis, Aspergillus fumigatus, Aspergillus niger, Botrytiscinerea, Candida dublinensis, Candida glabrata, Candida krusei, Candiaparapsilopsis, Candia tropicalis, Coccidioides immitis, Cryptococcusneoformans, Erysiphe graminis, Exophalia dermatiditis, Fusariumosysproum, Histoplasma capsulatum, Magnaporthe grisea, Mucor rouxii,Pneumocystis carinii, Puccinia graminis, Puccinia recodita, Rhizomucorpusillus, Puccinia striifommis, Rhizopus arrhizus, Septoria avenae,Septoria nodorum, Septoria triticii, Tilletia controversa, Tilletiatritici, Trichospoon beigelii and Ustilago maydis fumigatus.

Particularly preferred homologues of the present invention are fromAspergillus fumigatus. Particularly preferred homologous have an aminoacid sequence comprising SEQ ID NO: 48 to SEQ ID NO: 73.

Desirably, a homologue can be derived by using one or more of thedisclosed sequences to define a pair of primers to isolate thehomologue-encoding nucleic acid molecules from any desired species. Suchmolecules can be expressed to yield protein homologues by recombinantmeans.

A homologue of an essential polynucleotide polypeptide is a polypeptidehaving an amino acid sequence that is homologous to a natural essentialpolynucleotide polypeptide amino acid sequence that a nucleic acidsequence encoding the homologue is capable of hybridizing under reducedand/or high stringent conditions to a nucleic acid sequence encoding thenatural essential polynucleotide polypeptide amino acid sequencedisclosed herein. Preferably the homologue retains one or morebiological activities of essential polynucleotide.

Essential polynucleotide protein homologues of the invention includeallelic variations of the natural polynucleotide encoding the essentialpolynucleotide protein. A “natural” polynucleotide is one that is foundin nature. Essential polynucleotide protein homologues can be producedusing techniques known in the art, including but not limited to directmodifications to a polynucleotide encoding a protein using, for example,classic or recombinant DNA techniques to effect random or targetedmutapolynucleotidesis.

The present invention encompasses the essential polynucleotide proteinsthat have undergone posttranslational modification. Such modificationcan include, for example, glycosylation (e.g., including the addition ofN-linked and/or O-lined oligosaccharides) or post translationconformation changes or post translation deletions.

The present invention is also directed to polypeptides lacking a startmethionine. It is well known in the art that the N-terminal methionineencoded by the translation initiation codon generally is removed withhigh efficiency from any protein after translation in all eukaryoticcells. While the N-terminal methionine on most proteins also isefficiently removed in most prokaryotes, for some proteins, thisprokaryotic removal process is inefficient, depending on the nature ofthe amino acid to which the N-terminal methionine is covalently linked.

In specific embodiments, the present invention is directed to thefollowing polypeptides which correspond to fungal essential polypeptidesof the present invention lacking a start methionine: amino acids 2 to346 of SEQ ID NO:12 (CaYLR100w); amino acids 2 to 622 of SEQ ID NO:13(CaYDR341c); amino acids 2 to 255 of SEQ ID NO:14 (CaYLR022c); aminoacids 2 to 292 of SEQ ID NO:15 (CaYOL077c); amino acids 2 to 1042 of SEQID NO:16 (CaYNL132w); amino acids 2 to 750 of SEQ ID NO:17 (CaYGR145w);amino acids 2 to 268 of SEQ ID NO:18 (CaYDR412w); amino acids 2 to 371of SEQ ID NO:19 (CaYOL010w); amino acids 2 to 257 of SEQ ID NO:20(CaYOR004w); amino acids 2 to 466 of SEQ ID NO:21 (CaYOR056c); and/oramino acids 2 to 195 of SEQ ID NO:22 (CaYLR009w).

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the protein without substantial loss of biologicalfunction. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988(1993), reported variant KGF proteins having heparin binding activityeven after deleting 3, 8, or 27 amino-terminal amino acid residues.Similarly, Interferon gamma exhibited up to ten times higher activityafter deleting 8-10 amino acid residues from the carboxy terminus ofthis protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” In fact, only 23 unique amino acid sequences, outof more than 3,500 nucleotide sequences examined, produced a proteinthat significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N- or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptideof the present invention may, in fact, result in a significant increasein one or more of the biological activities of the polypeptide(s). Forexample, biological activity of many polypeptides are governed by thepresence of regulatory domains at either one or both termini. Suchregulatory domains effectively inhibit the biological activity of suchpolypeptides in lieu of an activation event (e.g., binding to a cognateligand or receptor, phosphorylation, proteolytic processing, etc.).Thus, by eliminating the regulatory domain of a polypeptide, thepolypeptide may effectively be rendered biologically active in theabsence of an activation event.

Features of the Polypeptide Encoded by Polynucleotide No:1

The polynucleotide sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:12) of the novel fungal essential gene, CaYLR100w(also referred to as FCG5), of the present invention. The CaYLR100wpolypeptide (SEQ ID NO:12) is encoded by nucleotides 1 to 1038 of SEQ IDNO:1 and has a predicted molecular weight of 39.0 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYLR100w. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 1038 of SEQ ID NO:1, and the polypeptide corresponding to aminoacids 2 thru 346 of SEQ ID NO:13. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

As illustrated in FIGS. 1 and 2 and described elsewhere herein, the C.albicans CaYLR100w (FCG5) polynucleotide of the present invention, hasbeen found to share 60% identity at the protein level with the S.cerevisiae ERG27 (“ScERG27”) polynucleotide which encodes 3-keto sterolreductase involved in ergosterol biosynthesis. The CaYLR100w has beendemonstrated biochemically to represent a 3-keto sterol reductaseinvolved in C-4 sterol demethylation.

Briefly, CaYLR100w was determined to represent a homolog of ScERG27 byusing a genetically modified strain where the target CaYLR100wpolynucleotide was placed under the control of the CaMET3 promoter.Using this system, reduced synthetic activity of the ergosterol pathwaywas observed upon reduced CaYLR100w expression via down-regulation ofthe CaMET3 promoter by methionine and cysteine (see FIG. 22B). A similareffect was also observed with a known polynucleotide CaERG1 (encodingsqualene epoxidase) used as a control (see FIG. 22A).

As shown in FIG. 22A, where a control strain caerg1Δ/PMET3-CaERG1 wasused (see FIG. 22A), downregulation of the CaERG1 promoter in thepresence of methionine and cysteine decreased incorporation of[¹⁴C]-acetate into the ergosterol biosynthetic pathway. The observeddecrease, ranging from 62 to 88% compared with untreated cells (FIG.24A), in incorporation of [¹⁴C]-acetate into ergosterol was observedbeginning within 1 hour of methionine and cysteine addition andcontinued to decrease over the 4.5 hour experimental period (FIG. 22A).Downregulation of CaYLR100w also had a significant effect on cell growthas demonstrated by decreased absorbance at OD₆₀₀ (FIG. 23A). However,the decrease in ergosterol synthesis by methionine and cysteine was notthe result of having fewer cells present compared with untreated cellsover the 4.5 hour time frame since all samples were adjusted to the samedensity prior to addition of radolabeled actetate.

Terbinofine is a specific inhibitor of CaErg1 (squalene epoxidase) andtherefore is capable of blocking ergosterol synthesis at squalene in thebiosynthetic pathway. The caerg1Δ/P_(MET3-)CaERG1 strain treated withterbinofine demonstrated a block in ergosterol biosynthesis at squaleneas can be visualized in lanes 1 and 2 in FIG. 22A. Downregulation of theergosterol biosynthetic pathway in the caerg1Δ/P_(MET3-)CaERG1 strainwith methionine and cysteine resulted in the decreased accumulation of[¹⁴C]-acetate into squalene as compared with the untreated cells (FIG.22A, lanes 5 and 6).

Results obtained with the test strain fcg5Δ/P_(MET3-)FCG5 were similarto those obtained with the caerg1Δ/P_(MET3-)CaERG1 control strain asseen in FIG. 22B and FIG. 23B. [¹⁴C]-acetate counts incorporated intothe ergosterol and lanosterol intermediates decreased between 62 to 88%compared with untreated cells following the 45 minute to 4.5 hourdownregulation period (FIGS. 22B and 24B). Cell growth of this strainwas also retarded in the presence of methionine and cysteine, asexpected (FIG. 23B). As seen with the caerg1Δ/P_(MET3-)CaERG1 controlstrain, terbinofine treatment blocked the incorporation of radiolabelledacetate into ergosterol, with a majority of the counts trapped insqualene. Methionine and cysteine treated fcg5Δ/P_(MET3-)FCG5 cells thatwere subsequently treated with terbinofine prior to the incorporation of[¹⁴C]-acetate demonstrated decreased counts associated with squalene ascompared with non-downregulated, terbinofine treated cells (FIG. 22B,lanes 5 and 6). These results demonstrate that downregulation of eitherthe CaERG1 or the CaFCG5 genes will result in decreased activity of theergosterol biosynthetic pathway in general.

Given its high homology with the S. cerevisiae counterpart, CaYLR100w orFCG5, in conjunction with the biochemical data provided herein, istherefore an otholog of ScERG27 that encodes 3-keto sterol reductase inC. albicans.

The CaYLR100w (FCG5) has been formally renamed “CaERG27”. As CaERG27 isan essential polynucleotide in C. albicans, downregulation withmethionine and cysteine would presumably render cells more susceptibleto inhibition by antifungal agents and thus would be extremely useful indrug discovery for fungal therapeutics.

CaYLR100w polynucleotides and polypeptides, including fragments andmodualtors thereof, are useful for the treatment, amelioration, and/ordetection of fungal diseases and/or disorders, and are also useful indrug discovery for identifying additional fungal therapeutics.

The invention also encompasses N- and/or C-terminal deletions of theCaYLR100w polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYLR100w deletionpolypeptides are encompassed by the present invention: M1-P346, S2-P346,L3-P346, L4-P346, K5-P346, D6-P346, S7-P346, T8-P346, V9-P346, A10-P346,V11-P346, I12-P346, T13-P346, G14-P346, T15-P346, S16-P346, S17-P346,N18-P346, L19-P346, G20-P346, F21-P346, N22-P346, I23-P346, A24-P346,V25-P346, R26-P346, L27-P346, L28-P346, E29-P346, G30-P346, L31-P346,P32-P346, D33-P346, N34-P346, K35-P346, E36-P346, I37-P346, T38-P346,L39-P346, V40-P346, V41-P346, T42-P346, S43-P346, R44-P346, T45-P346,L46-P346, P47-P346, K48-P346, V49-P346, K50-P346, E51-P346, V52-P346,I53-P346, S54-P346, D55-P346, I56-P346, K57-P346, K58-P346, Y59-P346,I60-P346, V61-P346, A62-P346, K63-P346, I64-P346, P65-P346, T66-P346,K67-P346, V68-P346, N69-P346, K70-P346, V71-P346, E72-P346, F73-P346,D74-P346, Y75-P346, L76-P346, L77-P346, V78-P346, D79-P346, F80-P346,T81-P346, D82-P346, M83-P346, V84-P346, S85-P346, I86-P346, L87-P346,S88-P346, A89-P346, Y90-P346, Y91-P346, E92-P346, L93-P346, N94-P346,K95-P346, R96-P346, Y97-P346, K98-P346, H99-P346, I100-P346, D101-P346,Y102-P346, L103-P346, F104-P346, I105-P346, N106-P346, A107-P346,A108-P346, Q109-P346, G110-P346, V111-P346, Y112-P346, G113-P346,G114-P346, I115-P346, D116-P346, W117-P346, T118-P346, G119-P346,A120-P346, V121-P346, L122-P346, E123-P346, V124-P346, L125-P346,Q126-P346, S127-P346, P128-P346, I129-P346, E130-P346, A131-P346,V132-P346, T133-P346, N134-P346, P135-P346, T136-P346, Y137-P346,K138-P346, L139-P346, Q140-P346, K141-P346, V142-P346, G143-P346,V144-P346, E145-P346, S146-P346, G147-P346, D148-P346, K149-P346,L150-P346, G151-P346, L152-P346, V153-P346, F154-P346, Q155-P346,A156-P346, N157-P346, V158-P346, F159-P346, G160-P346, P161-P346,Y162-P346, Y163-P346, F164-P346, I165-P346, H166-P346, R167-P346,I168-P346, K169-P346, H170-P346, L171-P346, L172-P346, E173-P346,N174-P346, G175-P346, G176-P346, K177-P346, I178-P346, V179-P346,W180-P346, V181-P346, S182-P346, S183-P346, L184-P346, M185-P346,S186-P346, S187-P346, P188-P346, K189-P346, Y190-P346, L191-P346,S192-P346, F193-P346, N194-P346, D195-P346, L196-P346, Q197-P346,L198-P346, L199-P346, R200-P346, S201-P346, P202-P346, A203-P346,S204-P346, Y205-P346, E206-P346, G207-P346, S208-P346, K209-P346,R210-P346, L211-P346, V212-P346, D213-P346, L214-P346, M215-P346,H216-P346, F217-P346, G218-P346, T219-P346, Y220-P346, N221-P346,K222-P346, L223-P346, E224-P346, R225-P346, E226-P346, H227-P346,G228-P346, I229-P346, K230-P346, Q231-P346, Y232-P346, L233-P346,V234-P346, H235-P346, P236-P346, G237-P346, I238-P346, F239-P346,T240-P346, S241-P346, F242-P346, S243-P346, F244-P346, F245-P346,Q246-P346, Y247-P346, L248-P346, N249-P346, V250-P346, F251-P346,T252-P346, Y253-P346, Y254-P346, G255-P346, M256-P346, L257-P346,F258-P346, L259-P346, F260-P346, Y261-P346, L262-P346, A263-P346,R264-P346, F265-P346, L266-P346, G267-P346, S268-P346, P269-P346,Y270-P346, H271-P346, N272-P346, I273-P346, S274-P346, G275-P346,Y276-P346, I277-P346, A278-P346, A279-P346, N280-P346, A281-P346,P282-P346, V283-P346, A284-P346, A285-P346, A286-P346, L287-P346,G288-P346, Q289-P346, T290-P346, K291-P346, Q292-P346, N293-P346,C294-P346, K295-P346, T296-P346, A297-P346, S298-P346, A299-P346,C300-P346, T301-P346, R302-P346, S303-P346, G304-P346, K305-P346,E306-P346, Y307-P346, L308-P346, L309-P346, E310-P346, E311-P346,E312-P346, I313-P346, D314-P346, S315-P346, T316-P346, G317-P346,L318-P346, D319-P346, D320-P346, V321-P346, V322-P346, L323-P346,Y324-P346, L325-P346, D326-P346, T327-P346, L328-P346, T329-P346,K330-P346, E331-P346, W332-P346, D333-P346, E334-P346, K335-P346,L336-P346, K337-P346, D338-P346, Q339-P346, and/or 1340-P346 of SEQ IDNO:12. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseN-terminal CaYLR100w deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYLR100w deletionpolypeptides are encompassed by the present invention: M1-P346, M1-Q345,M1-R344, M1-T343, M1-N342, M1-V341, M1-I340, M1-Q339, M1-D338, M1-K337,M1-L336, M1-K335, M1-E334, M1-D333, M1-W332, M1-E331, M1-K330, M1-T329,M1-L328, M1-T327, M1-D326, M1-L325, M1-Y324, M1-L323, M1-V322, M1-V321,M1-D320, M1-D319, M1-L318, M1-G317, M1-T316, M1-S315, M1-D314, M1-I313,M1-E312, M1-E311, M1-E310, M1-L309, M1-L308, M1-Y307, M1-E306, M1-K305,M1-G304, M1-S303, M1-R302, M1-T301, M1-C300, M1-A299, M1-S298, M1-A297,M1-T296, M1-K295, M1-C294, M1-N293, M1-Q292, M1-K291, M1-T290, M1-Q289,M1-G288, M1-L287, M1-A286, M1-A285, M1-A284, M1-V283, M1-P282, M1-A281,M1-N280, M1-A279, M1-A278, M1-I277, M1-Y276, M1-G275, M1-S274, M1-I273,M1-N272, M1-H271, M1-Y270, M1-P269, M1-S268, M1-G267, M1-L266, M1-F265,M1-R264, M1-A263, M1-L262, M1-Y261, M1-F260, M1-L259, M1-F258, M1-L257,M1-M256, M1-G255, M1-Y254, M1-Y253, M1-T252, M1-F251, M1-V250, M1-N249,M1-L248, M1-Y247, M1-Q246, M1-F245, M1-F244, M1-S243, M1-F242, M1-S241,M1-T240, M1-F239, M1-I238, M1-G237, M1-P236, M1-H235, M1-V234, M1-L233,M1-Y232, M1-Q231, M1-K230, M1-I229, M1-G228, M1-H227, M1-E226, M1-R225,M1-E224, M1-L223, M1-K222, M1-N221, M1-Y220, M1-T219, M1-G218, M1-F217,M1-H216, M1-M215, M1-L214, M1-D213, M1-V212, M1-L211, M1-R210, M1-K209,M1-S208, M1-G207, M1-E206, M1-Y205, M1-S204, M1-A203, M1-P202, M1-S201,M1-R200, M1-L199, M1-L198, M1-Q197, M1-L196, M1-D195, M1-N194, M1-F193,M1-S192, M1-L191, M1-Y190, M1-K189, M1-P188, M1-S187, M1-S186, M1-M185,M1-L184, M1-S183, M1-S182, M1-V181, M1-W180, M1-V179, M1-I178, M1-K177,M1-G176, M1-G175, M1-N174, M1-E173, M1-L172, M1-L171, M1-H170, M1-K169,M1-I168, M1-R167, M1-H166, M1-I165, M1-F164, M1-Y163, M1-Y162, M1-P161,M1-G160, M1-F159, M1-V158, M1-N157, M1-A156, M1-Q155, M1-F154, M1-V153,M1-L152, M1-G151, M1-L150, M1-K149, M1-D148, M1-G147, M1-S146, M1-E145,M1-V144, M1-G143, M1-V142, M1-K141, M1-Q140, M1-L139, M1-K138, M1-Y137,M1-T136, M1-P135, M1-N134, M1-T133, M1-V132, M1-A131, M1-E130, M1-I129,M1-P128, M1-S127, M1-Q126, M1-L125, M1-V124, M1-E123, M1-L122, M1-V121,M1-A120, M1-G119, M1-T118, M1-W117, M1-D116, M1-I115, M1-G114, M1-G113,M1-Y112, M1-V111, M1-G110, M1-Q109, M1-A108, M1-A107, M1-N106, M1-I105,M1-F104, M1-L103, M1-Y102, M1-D101, M1-I100, M1-H99, M1-K98, M1-Y97,M1-R96, M1-K95, M1-N94, M1-L93, M1-E92, M1-Y91, M1-Y90, M1-A89, M1-S88,M1-L87, M1-I86, M1-S85, M1-V84, M1-M83, M1-D82, M1-T81, M1-F80, M1-D79,M1-V78, M1-L77, M1-L76, M1-Y75, M1-D74, M1-F73, M1-E72, M1-V71, M1-K70,M1-N69, M1-V68, M1-K67, M1-T66, M1-P65, M1-I64, M1-K63, M1-A62, M1-V61,M1-I60, M1-Y59, M1-K58, M1-K57, M1-I56, M1-D55, M1-S54, M1-I53, M1-V52,M1-E51, M1-K50, M1-V49, M1-K48, M1-P47, M1-L46, M1-T45, M1-R44, M1-S43,M1-T42, M1-V41, M1-V40, M1-L39, M1-T38, M1-I37, M1-E36, M1-K35, M1-N34,M1-D33, M1-P32, M1-L31, M1-G30, M1-E29, M1-L28, M1-L27, M1-R26, M1-V25,M1-A24, M1-I23, M1-N22, M1-F21, M1-G20, M1-L19, M1-N18, M1-S17, M1-S16,M1-T15, M1-G14, M1-T13, M1-I12, M1-V11, M1-A10, M1-V9, M1-T8, and/orM1-S7 of SEQ ID NO:12. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal CaYLR100w deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polynucleotide sequence provided in SEQ ID NO:1, andin particular to the coding region of the polynucleotide sequenceprovided in SEQ ID NO:1. Preferably such polynucleotides encodepolypeptides that have biological activity, particularly 3-keto sterolreductase activity.

The present invention also encompasses polypeptides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polypeptide sequence provided in SEQ ID NO:12.

Most preferred are polypeptides that share at least about 99.5% identitywith the polypeptide sequence provided in SEQ ID NO:12.

The present invention is also directed to a homology model detailing thethree-dimensional structure of the CaYLR100w polypeptide (SEQ ID NO:12)of the present invention.

Protein threading and molecular modeling of CaYLR100w suggest thatCaYLR100w has a three dimensional fold similar to that of the porcinecarbonyl reductase EC number 1.1.1.-(Ghosh et al., 2001), Protein DataBank (PDB, Bernstein et. al., 1977 & Berman et. al., 2000) entry 1HU4.Based on sequence, structure, motifs and known short chaindehydrogenase/reductase family signature sequences, CaYLR100w contains anovel known short chain dehydrogenase/reductase domain found also in3-keto sterol reductases.

The polypeptide CaYLR100w contains a distinct structural domain know asthe short chain dehydrogenase/reductase (SDR) superfamily catalyticdomain which contains the active site. The three dimensionalcrystallographic structure for several short chaindehydrogenase/reductases have been reported and are deposited into theProtein Data Bank (Ghosh et al., 2001, 2000, Bernstein et. al., 1977,Berman et. al., 2000). The structure (Protein Data Bank, PDB entry 1HU4)of the carbonyl reductase from pig (porcine) is similar to the othershort chain dehydrogenase/reductases (EC 1.1.1.-) and is the closeststructural homolog of CaYLR100w.

The short chain dehydrogenase/reductase (SDR) family is a very largefamily of enzymes that are known to be NAD- or NADP-dependentoxidoreductases. Most members of this family are 250 to 300 amino acidsin length. This family of proteins uses a Tyr-Lys-Ser triad as catalyticresidues. The SDRs catalyze the activation and inactivation of steroids,vitamins, protstaglandins and other bioactive molecules by oxidation andreduction of hydroxyl and carbonyl groups, respectively. CaYLR100w isthought to have the 3-keto sterol reductase activity and the 3-ketosterol reductases are members of the SDR superfamily.

The basic SDR fold includes a seven stranded parallel beta sheet flankedby three parallel helices on each side. The core of this domain containsthe classic “Rossman fold” that has been associated with coenzyme NADPHbinding. Mutagenesis and modeling experiments have suggested that a Tyrand Lys (part of a catalytic triad) comprising the YXXXK (SEQ ID NO:254)motif, demonstrate that the Tyr proton as a donor in electrophilicattack on the substrate carbonyl in a reduction reaction. These SDRs arealso referred to as short chain dehydrogenase/reductases and seem toshare the same core domain tertiary structure based on a Rossmann fold.

This structure-based information and sequence information from novelgenes can be used to identify other protein family members that sharethis same fold.

The present invention provides a three dimensional model of theCaYLR100w polypeptide. The three dimensional model provides for aspecific description of the catalytic core and functional sites in the3-keto sterol CaYLR100w polypeptide.

The catalytic core and functional sites are defined by atomiccoordinates (Table 8). Based on these data, the inventors have ascribedthe CaYLR100w polypeptide as having dehydrogenase/reductase activity(s),specifically the 3-keto sterol reductase activity and cellular andsystemic regulatory function(s). Specifically the reductase activityrelates to the activation and/or inactivation of steroids, vitamins,protstaglandins and other bioactive molecules by reduction of hydroxylgroups. For CaYLR100w it is the reductase activity at the 3-ketoposition of steroids during the biosynthesis of ergosterol.

Homology models are useful when there is no experimental informationavailable on the protein of interest. A three dimensional model can beconstructed on the basis of the known structure of a homologous protein(Greer et. al., 1991, Lesk, et. al., 1992, Levitt, 1992, Cardozo, et.al., 1995, Sali, et. al., 1995).

Those of skill in the art will understand that a homology model isconstructed on the basis of first identifying a template, or, protein ofknown structure which is similar to the protein without known structure.This can be accomplished by pairwise alignment of sequences using suchprograms as FASTA (Pearson, et. al. 1990) and BLAST (Altschul, et. al.,1990). In cases where sequence similarity is high (greater than 30%)these pairwise comparison methods may be adequate. Likewise, multiplesequence alignments or profile-based methods can be used to align aquery sequence to an alignment of multiple (structurally andbiochemically) related proteins. When the sequence similarity is low,more advanced techniques are used such as fold recognition (proteinthreading; Hendlich, et. al., 1990, Koppensteiner et. Al. 2000, Sippl &Weitckus 1992, Sippl 1993), where the compatibility of a particularsequence with the three dimensional fold of a potential template proteinis gauged on the basis of a knowledge-based potential. Following theinitial sequence alignment, the query template can be optimally alignedby manual manipulation or by incorporation of other features (motifs,secondary structure predictions, and allowed sequence conservation,etc.). Next, structurally conserved regions can be identified and areused to construct the core secondary structure (Levitt, 1992, Sali, et.al., 1995) elements in the three dimensional model. Variable regions,called “unconserved regions” and loops can be added usingknowledge-based techniques. The complete model with variable regions andloops can be refined performing forcefield calculations (Sali, et. al.,1995, Cardozo, et. al., 1995).

For CaYLR100w, a multiple sequence alignment generated manually bycombining results from protein threading pairwise alignments and thesepairwise alignments were used to align the sequence of CaYLR100w withthe sequence of porcine carbonyl reductase, alcohol dehydrogenases, 17βhydroxysteroid dehydrogenases and other SDRs for which three dimensionalstructures exist. The alignment produced a sequence identity of 20%between the porcine carbonyl reductase (Gosh et. al. 2001, Protein DataBank entry 1HU4; Genbank Accession No. gil15826210; SEQ ID NO:251). Thealignment of CaYLR100w with PDB entry 1HU4 chain A is set forth in FIG.26.

For the present invention, the homology model of CaYLR100w was derivedfrom the sequence alignment set forth in FIG. 26. An overall atomicmodel including plausible sidechain orientations was generated using theprogram LOOK (Levitt, 1992). The three dimensional model for CaYLR100wis defined by the set of structure coordinates as set forth in Table 8and is shown in FIG. 27 rendered by backbone secondary structures.

In order to recognize errors in three-dimensional structures, knowledgebased mean fields can be used to judge the quality of protein folds(Sippl 1993). The methods can be used to recognize misfolded structuresas well as faulty parts of structural models. The technique generates anenergy graph where the energy distribution for a given protein fold isdisplayed on the y-axis and residue position in the protein fold isdisplayed on the x-axis. The knowledge based mean fields compose a forcefield derived from a set of globular protein structures taken as asubset from the Protein Data Bank (Bernstein et. al. 1977). To analyzethe quality of a model, the energy distribution is plotted and comparedto the energy distribution of the template from which the model wasgenerated. FIG. 28 shows the energy graph for the CaYLR100w model(dotted line) and the template (porcine carbonyl reductase) from whichthe model was generated. The model has virtually an identical energyplot when compared to the short chain dehydrogenase/reductase templatedemonstrating that CaYLR100w has similar structural characteristicsexcept for one region corresponding to residues 100-150 of CaYLR100w.However the energy plot suggests the overall model three-dimensionalfold for CaYLR100w is “native-like”. This graph supports the motif andsequence alignments described herein in confirming that the threedimensional structure coordinates of CaYLR100w are an accurate anduseful representation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model.

Those of skill in the art will understand that a set of structurecoordinates for a protein is a relative set of points that define ashape in three dimensions. Thus, it is possible that an entirelydifferent set of coordinates could define a similar or identical shape.Moreover, slight variations in the individual coordinates, as emanatefrom generation of similar homology models using different alignmenttemplates (i.e., other than the structure coordinates of 1HU4), and/orusing different methods in generating the homology model, will haveminor effects on the overall shape. Variations in coordinates may alsobe generated because of mathematical manipulations of the structurecoordinates. For example, the structure coordinates set forth in Table 8could be manipulated by fractionalization of the structure coordinates;integer additions or subtractions to sets of the structure coordinates,inversion of the structure coordinates or any combination of the above.

Various computational analyses are therefore necessary to determinewhether a molecule or a portion thereof is sufficiently similar to allor parts of CaYLR100w described above as to be considered the same. Suchanalyses may be carried out in current software applications, such asINSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as describedin the User's Guide, online or software applications available in theSYBYL software suite (Tripos Inc., St. Louis, Mo.).

Using the superimposition tool in the program INSIGHTII comparisons canbe made between different structures and different conformations of thesame structure. The procedure used in INSIGHTII to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. Since atom equivalency within INSIGHTII isdefined by user input, for the purpose of this invention equivalentatoms are defined as protein backbone atoms (N, Cα, C and O) for allconserved residues between the two structures being compared. Also, onlyrigid fitting operations were considered. When a rigid fitting method isused, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atom is anabsolute minimum. This number, given in angstroms, is reported byINSIGHTII.

For the purpose of this invention, any homology model of a CaYLR100wthat has a root mean square deviation of conserved residue backboneatoms (N, Cα, C, O) of less than about 2.0 A when superimposed on therelevant backbone atoms described by structure coordinates listed inTable 8 are considered identical. More preferably, the root mean squaredeviation is less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, or 0.1 Angstroms

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a protein from the relevant portion of thebackbone of CaYLR100w defined by the structure coordinates describedherein.

This invention as embodied by the three-dimensional model enables thestructure-based design of modulators of the biological function ofCaYLR100w, as well as mutants with altered biological function and/orspecificity.

The sequence alignment (FIG. 26) used as a template for creating thethree-dimensional model of CaYLR100w short chain dehydrogenase/reductasedomain shows 20% sequence identity between catalytic domain of CaYLR100wand porcine carbonyl reductase, PDB code 1HU4.

In the active site of short chain dehydrogenase/reductases (“SDRS”),there is a catalytic triad (YKS) that catalyzes the activation andinactivation of steroids, vitamins, protstaglandins and other bioactivemolecules by oxidation and reduction of hydroxyl and carbonyl groups,respectively. The tyrosine side chain is thought to act as the protondonor in electrophilic attack on the substrate carbonyl in a reductionreaction. In porcine carbonyl reductase, the catalytic triad consists ofY193, K197 and S139. In the model and alignment, FIG. 26 and FIG. 27 ofCaYLR100w shows that two of the three catalytic residues are conservedand are displayed in the active site. The tyrosine and serine areconserved in CaYLR100w (Y247 and S183) but the lysine position is aphenylalanine (F251) in CaYLR100w. The fact that two of the threecatalytic residues are conserved supports the assignment of function forCaYLR100w as a 3-keto sterol reductase member of the SDR superfamily.

The conservation of the catalytic amino acids as part of the active siteand the overall 20% sequence identity emphasizes the significance of thethree-dimensional model of the CaYLR100w polypeptide. The conservedresidues are located in the functional sites that are essential forcoenzyme and substrate binding. These active site residues play criticalroles in the mechanism of catalysis, substrate specificity, and coenzymebinding.

The structure coordinates of the CaYLR100w homology model, portionthereofs, are preferably stored in a machine-readable storage medium.Such data may be used for a variety of purposes, such as drug discoveryand target prioritization and validation.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table 8.

For the first time, the present invention permits the use, throughhomology modeling based upon the sequence of CaYLR100w (FIGS. 26 and 27)of structure-based or rational drug design techniques to design, select,and synthesizes chemical entities that are capable of modulating thebiological function of CaYLR100w. Comparison of the CaYLR100w homologymodel with the structures of other SDRs enable the use of rational orstructure based drug design methods to design, select or synthesizespecific chemical modulators of CaYLR100w.

Accordingly, the present invention is also directed to the entiresequence in FIG. 11, or any portion thereof for the purpose ofgenerating a homology model for the purpose of three dimensionalstructure-based drug designs.

The present invention also encompasses mutants or homologues of thesequence in FIG. 11, or any portion thereof. In a preferred embodiment,the mutants or homologues have at least 25% identity, more preferably50% identity, more preferably 75% identity, and most preferably 90%identity to the amino acid residues in FIG. 11 (SEQ ID NO:12).

The three-dimensional model structure of the CaYLR100w will also providemethods for identifying modulators of biological function. Variousmethods or combination thereof can be used to identify these compounds.

Structure coordinates of the active site region defined above can alsobe used to identify structural and chemical features. Identifiedstructural or chemical features can then be employed to design or selectcompounds as potential CaYLR100w modulators. By structural and chemicalfeatures it is meant to include, but is not limited to, van der Waalsinteractions, hydrogen bonding interactions, charge interaction,hydrophobic interactions, and dipole interaction. Alternatively, or inconjunction, the three-dimensional structural model can be employed todesign or select compounds as potential CaYLR100w modulators. Compoundsidentified as potential CaYLR100w modulators can then be synthesized andscreened in an assay characterized by binding of a test compound to theCaYLR100w, or in characterizing CaYLR100w deactivation in the presenceof a small molecule. Examples of assays useful in screening of potentialCaYLR100w modulators include, but are not limited to, screening insilico, in vitro assays and high throughput assays. Finally, thesemethods may also involve modifying or replacing one or more amino acidsfrom CaYLR100w according to Table 8.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number of computer modeling systems are available inwhich the sequence of the CaYLR100w and the CaYLR100w structure (i.e.,atomic coordinates of CaYLR100w and/or the atomic coordinates of theactive site region as provided in Table 8) can be input. The computersystem then generates the structural details of one or more theseregions in which a potential CaYLR100w modulator binds so thatcomplementary structural details of the potential modulators can bedetermined. Design in these modeling systems is generally based upon thecompound being capable of physically and structurally associating withCaYLR100w. In addition, the compound must be able to assume aconformation that allows it to associate with CaYLR100w. Some modelingsystems estimate the potential inhibitory or binding effect of apotential CaYLR100w modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are well known. Often thesemethods begin by visual inspection of the binding site on the computerscreen. Selected fragments or chemical entities are then positioned inone or more positions and orientations within the active site region inCaYLR100w. Molecular docking is accomplished using software such asINSIGHTII, ICM (Molsoft LLC, La Jolla, Calif.), and SYBYL, following byenergy minimization and molecular dynamics with standard molecularmechanic forcefields such as CHARMM and MMFF. Examples of computerprograms which assist in the selection of chemical fragment or chemicalentities useful in the present invention include, but are not limitedto, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntzet. al. 1982).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992), LeapFrog (Tripos Associates, St. Louis Mo.) and DOCK (Kuntzet. al., 1982). Programs such as DOCK (Kuntz et. al. 1982) can be usedwith the atomic coordinates from the homology model to identifypotential ligands from databases or virtual databases which potentiallybind the in the active site region, and which may therefore be suitablecandidates for synthesis and testing. The computer programs may utilizea combination of the following steps:

1) Selection of fragments or chemical entities from a database and thenpositioning the chemical entity in one or more orientations within theCaYLR100w catalytic domain defined by Table 8

2) Characterization of the structural and chemical features of thechemical entity and active site including van der Waals interactions,hydrogen bonding interactions, charge interaction, hydrophobic bondinginteraction, and dipole interactions

3) Search databases for molecular fragments which can be joined to orreplace the docked chemical entity and spatially fit into regionsdefined by the said CaYLR100w catalytic domain or catalytic domainactive/functional sites

4) Evaluate the docked chemical entity and fragments using a combinationof scoring schemes which account for van der Waals interactions,hydrogen bonding interactions, charge interaction, hydrophobicinteractions

Databases that may be used include ACD (Molecular Designs Limited),Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute),Maybridge (Maybridge Chemical Company Ltd), CCDC (CambridgeCrystallographic Data Center), CAST (Chemical Abstract Service), Derwent(Derwent Information Limited).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and CaYLR100w, can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm1992) as well as 3D Database systems (Martin 1992).

Additionally, the three-dimensional homology model of CaYLR100w will aidin the design of mutants with altered biological activity. Site directedmutagenesis can be used to generate proteins with similar or varyingdegrees of biological activity compared to native CaYLR100w. Thisinvention also relates to the generation of mutants or homologs ofCaYLR100w. It is clear that molecular modeling using the threedimensional structure coordinates set forth in Table 8 and visualizationof the CaYLR100w model, FIG. 27 and alignment in FIG. 26 can be utilizedto design homologs or mutant polypeptides of CaYLR100w that have similaror altered biological activities, function or reactivities.

The invention also relates to in silico screening methods including insilico docking and methods of structure based drug design which utilizethe three dimensional coordinates of CaYLR100w (Table 8). Also providedare methods of identifying modulators of CaYLR100w that includemodulator building or searching utilizing computer programs andalgorithms. In an embodiment of the invention a method is provided fordesigning potential modulators of CaYLR100w comprising any combinationof steps which utilize said three dimensional structure to design orselect potential modulators.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 1024 ofSEQ ID NO:1, b is an integer between 15 to 1038, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:1,and where b is greater than or equal to a+14

Features of the Polypeptide Encoded by Polynucleotide No:2

The polynucleotide sequence (SEQ ID NO:2) and deduced amino acidsequence (SEQ ID NO:13) of the novel fungal essential gene, CaYDR341c(also referred to as FCG6), of the present invention. The CaYDR341cpolypeptide (SEQ ID NO:13) is encoded by nucleotides 1 to 1866 of SEQ IDNO:2 and has a predicted molecular weight of 70.8 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYDR341c. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 1866 of SEQ ID NO:2, and the polypeptide corresponding to aminoacids 2 thru 622 of SEQ ID NO:14. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

As illustrated in FIGS. 1 and 2 and described elsewhere herein, theCaYDR341c polypeptide of the present invention shares 65% identity withS. cerevisiae CaYDR341c. Based upon homology to known proteins,CaYDR341c has been predicted to encode an arginine tRNA synthetaseinvolved in protein synthesis. Experiments described herein demonstratethat downregulation of a genetically manipulated strain having CaYDR341c(FCG6) placed under the control of the CaMET3 promoter with methionineand cysteine affects protein synthesis. As shown in FIG. 25,incorporation of radiolabelled leucine and arginine into charged tRNAsand protein was greatly reduced in the presence of methionine andcysteine after a 3.5 hour induction period as compared with strains inthe absence of methionine and cysteine.

Based upon homology to known arginine tRNA synthetases, in conjunctionwith the biochemical data shown herein, it is clear that CaYDR341c isinvolved in protein systhesis.

Briefly, the results illustrated in FIG. 25 clearly show a dramaticeffect of the 3.5 hour methionine/cysteine downregulation on generalprotein synthesis. This is demonstrated by the fact that arginineincorporation into polypeptide is almost completely inhibited (98.6%inhibition as seen in FIGS. 25A and 25B) in the presence of methionineand cysteine as compared with that in the absence of methionine andcysteine. Leucine incorporation is also greatly impaired by as much as96.6% with methionine and cysteine treated cells compared to untreatedcells (FIGS. 25A and 25B). This result is expected since a blockobtained by insufficient argininyl-tRNA would be expected to halt thefurther elongation of polypeptide synthesis, including the furtherincorporation of [³H]-leucine. Therefore, the results provide directbiochemical evidence that CaYDR341c is indeed involved in proteinsynthesis, and encodes an argininyl-tRNA synthetase.

CaYDR341c polynucleotides and polypeptides, including fragments andmodualtors thereof, are useful for the treatment, amelioration, and/ordetection of fungal diseases and/or disorders, and are also useful indrug discovery for identifying additional fungal therapeutics.

The invention also encompasses N- and/or C-terminal deletions of theCaYDR341c polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYDR341c deletionpolypeptides are encompassed by the present invention: M1-M622, S2-M622,V3-M622, E4-M622, T5-M622, I6-M622, S7-M622, D8-M622, S9-M622, L10-M622,K11-M622, Q12-M622, L13-M622, G14-M622, L15-M622, S16-M622, Q17-M622,P18-M622, A19-M622, A20-M622, I21-M622, E22-M622, G23-M622, T24-M622,H25-M622, P26-M622, Q27-M622, Y28-M622, N29-M622, V30-M622, V31-M622,D32-M622, V33-M622, F34-M622, R35-M622, N36-M622, Y37-M622, I38-M622,A39-M622, E40-M622, E41-M622, L42-M622, H43-M622, R44-M622, I45-M622,S46-M622, S47-M622, V48-M622, D49-M622, K50-M622, S51-M622, I52-M622,I53-M622, I54-M622, Q55-M622, A56-M622, L57-M622, D58-M622, T59-M622,P60-M622, K61-M622, V62-M622, L63-M622, D64-M622, Q65-M622, G66-M622,D67-M622, I68-M622, I69-M622, V70-M622, P71-M622, I72-M622, P73-M622,K74-M622, L75-M622, R76-M622, L77-M622, K78-M622, G79-M622, I80-M622,N81-M622, P82-M622, N83-M622, E84-M622, K85-M622, S86-M622, K87-M622,E88-M622, W89-M622, A90-M622, E91-M622, N92-M622, F93-M622, N94-M622,K95-M622, G96-M622, K97-M622, F98-M622, I99-M622, S100-M622, E01-M622,I102-M622, K103-M622, P104-M622, Q105-M622, G106-M622, V107-M622,F108-M622, L109-M622, Q110-M622, F111-M622, Y112-M622, F113-M622,A114-M622, K115-M622, T116-M622, L117-M622, L118-M622, Y119-M622,N120-M622, L121-M622, V122-M622, I123-M622, E124-M622, D125-M622,V126-M622, L127-M622, K128-M622, R129-M622, K130-M622, S131-M622,D132-M622, Y133-M622, G134-M622, Y135-M622, L136-M622, P137-M622,L138-M622, G139-M622, V140-M622, G141-M622, K142-M622, K143-M622, A144-M622, I145-M622, V146-M622, E147-M622, F148-M622, S149-M622,S150-M622, P151-M622, N152-M622, I153-M622, A154-M622, K155-M622,P156-M622, F157-M622, H158-M622, A159-M622, G160-M622, H161-M622,L162-M622, R163-M622, S164-M622, T165-M622, I166-M622, I167-M622,G168-M622, G169-M622, F170-M622, I171-M622, S1172-M622, N173-M622,L174-M622, Y175-M622, E176-M622, K177-M622, V178-M622, G179-M622,W180-M622, D181-M622, V182-M622, T183-M622, R184-M622, I185-M622,N186-M622, Y187-M622, L188-M622, G1189-M622, D190-M622, W191-M622,G192-M622, K193-M622, Q194-M622, F195-M622, G196-M622, L197-M622,L198-M622, A199-M622, V200-M622, G201-M622, F202-M622, E203-M622,R204-M622, Y205-M622, G206-M622, D207-M622, E208-M622, S209-M622,K210-M622, L211-M622, A212-M622, S213-M622, D214-M622, P215-M622,I216-M622, N217-M622, H218-M622, L219-M622, F220-M622, E221-M622,V222-M622, Y223-M622, V224-M622, K225-M622, I226-M622, N227-M622,Q228-M622, D229-M622, V230-M622, T231-M622, K232-M622, E233-M622,T234-M622, S235-M622, E236-M622, A237-M622, T238-M622, G239-M622,E240-M622, T241-M622, P242-M622, A243-M622, E244-M622, T245-M622,I246-M622, D247-M622, A248-M622, S249-M622, E250-M622, Q251-M622,D252-M622, E253-M622, K254-M622, K255-M622, I256-M622, Q257-M622,S258-M622, S259-M622, T260-M622, N261-M622, E262-M622, E263-M622,A264-M622, R265-M622, R266-M622, F267-M622, F268-M622, R269-M622,R270-M622, M271-M622, E272-M622, D273-M622, G274-M622, D275-M622,E276-M622, S277-M622, A278-M622, L279-M622, K280-M622, I281-M622,W282-M622, A283-M622, R284-M622, F285-M622, R286-M622, D287-M622,L288-M622, S289-M622, I290-M622, E291-M622, K292-M622, Y293-M622,V294-M622, D295-M622, T296-M622, Y297-M622, G298-M622, R299-M622,L300-M622, N301-M622, I302-M622, K303-M622, Y304-M622, D305-M622,V306-M622, Y307-M622, S308-M622, G309-M622, E310-M622, S311-M622,Q312-M622, V313-M622, P314-M622, Q315-M622, E316-M622, K317-M622,M318-M622, K319-M622, E320-M622, A321-M622, T322-M622, K323-M622,L324-M622, F325-M622, E326-M622, D327-M622, K328-M622, G329-M622,L330-M622, I331-M622, D332-M622, I333-M622, D334-M622, R335-M622,G336-M622, A337-M622, K338-M622, L339-M622, I340-M622, D341-M622,L342-M622, T343-M622, K344-M622, F345-M622, N346-M622, K347-M622,K348-M622, L349-M622, G350-M622, K351-M622, A352-M622, L353-M622,V354-M622, E355-M622, K356-M622, S357-M622, D358-M622, G359-M622,T360-M622, S361-M622, L362-M622, Y363-M622, L364-M622, T365-M622,R366-M622, D367-M622, V368-M622, G369-M622, E370-M622, A371-M622,I372-M622, K373-M622, R374-M622, Y375-M622, E376-M622, T377-M622,Y378-M622, K379-M622, F380-M622, D381-M622, K382-M622, M383-M622,I384-M622, Y385-M622, V386-M622, I387-M622, A388-M622, A389-M622,Q390-M622, Q391-M622, D392-M622, L393-M622, H394-M622, C395-M622,A396-M622, Q397-M622, F398-M622, F399-M622, E400-M622, I401-M622,L402-M622, K403-M622, Q404-M622, M405-M622, G406-M622, F407-M622,E408-M622, W409-M622, A410-M622, H411-M622, N412-M622, L413-M622,E414-M622, H415-M622, V416-M622, N417-M622, F418-M622, G419-M622,M420-M622, V421-M622, Q422-M622, G423-M622, M424-M622, S425-M622,T426-M622, R427-M622, K428-M622, G429-M622, T430-M622, V431-M622,V432-M622, F433-M622, L434-M622, D435-M622, N436-M622, I437-M622,L438-M622, Q439-M622, E440-M622, T441-M622, K442-M622, E443-M622,K444-M622, M445-M622, H446-M622, E447-M622, V448-M622, M449-M622,Q450-M622, K451-M622, N452-M622, E453-M622, E454-M622, K455-M622,Y456-M622, A457-M622, Q458-M622, I459-M622, E460-M622, D461-M622,P462-M622, D463-M622, K464-M622, I465-M622, A466-M622, D467-M622,L468-M622, I469-M622, G470-M622, I471-M622, S472-M622, A473-M622,V474-M622, M475-M622, I476-M622, Q477-M622, D478-M622, M479-M622,Q480-M622, S481-M622, K482-M622, R483-M622, I484-M622, H485-M622,N486-M622, Y487-M622, E488-M622, F489-M622, K490-M622, W491-M622,D492-M622, R493-M622, M494-M622, T495-M622, S496-M622, F497-M622,E498-M622, G499-M622, D500-M622, T501-M622, G502-M622, P503-M622,Y504-M622, L505-M622, Q506-M622, Y507-M622, A508-M622, H509-M622,S510-M622, R511-M622, L512-M622, C513-M622, S514-M622, M515-M622,Q516-M622, R517-M622, K518-M622, S519-M622, G520-M622, I521-M622,S522-M622, I523-M622, E524-M622, E525-M622, L526-M622, E527-M622,H528-M622, A529-M622, N530-M622, F531-M622, D532-M622, L533-M622,L534-M622, V535-M622, E536-M622, P537-M622, C538-M622, A539-M622,S540-M622, A541-M622, L542-M622, A543-M622, R544-M622, T545-M622,L546-M622, A547-M622, Q548-M622, Y549-M622, P550-M622, D551-M622,V552-M622, I553-M622, K554-M622, K555-M622, A556-M622, V557-M622,K558-M622, G559-M622, L560-M622, E561-M622, P562-M622, S563-M622,T564-M622, I565-M622, V566-M622, T567-M622, Y568-M622, L569-M622,F570-M622, S571-M622, V572-M622, T573-M622, H574-M622, I575-M622,V576-M622, S577-M622, Q578-M622, C579-M622, Y580-M622, D581-M622,I582-M622, L583-M622, W584-M622, V585-M622, S586-M622, G587-M622,Q588-M622, E589-M622, K590-M622, D591-M622, V592-M622, A593-M622,I594-M622, A595-M622, R596-M622, L597-M622, A598-M622, L599-M622,Y600-M622, E601-M622, A602-M622, A603-M622, R604-M622, Q605-M622,V606-M622, I607-M622, N608-M622, N609-M622, G610-M622, M611-M622,T612-M622, L613-M622, L614-M622, G615-M622, and/or L616-M622 of SEQ IDNO:13. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseN-terminal CaYDR341c deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYDR341c deletionpolypeptides are encompassed by the present invention: M1-M622, M1-R621,M1-N620, M1-V619, M1-P618, M1-T617, M1-L616, M1-G615, M1-L614, M1-L613,M1-T612, M1-M611, M1-G610, M1-N609, M1-N608, M1-I607, M1-V606, M1-Q605,M1-R604, M1-A603, M1-A602, M1-E601, M1-Y600, M1-L599, M1-A598, M1-L597,M1-R596, M1-A595, M1-I594, M1-A593, M1-V592, M1-D591, M1-K590, M1-E589,M1-Q588, M1-G587, M1-S586, M1-V585, M1-W584, M1-L583, M1-I582, M1-D581,M1-Y580, M1-C579, M1-Q578, M1-S577, M1-V576, M1-I575, M1-H574, M1-T573,M1-V572, M1-S571, M1-F570, M1-L569, M1-Y568, M1-T567, M1-V566, M1-I565,M1-T564, M1-S563, M1-P562, M1-E561, M1-L560, M1-G559, M1-K558, M1-V557,M1-A556, M1-K555, M1-K554, M1-I553, M1-V552, M1-D551, M1-P550, M1-Y549,M1-Q548, M1-A547, M1-L546, M1-T545, M1-R544, M1-A543, M1-L542, M1-A541,M1-S540, M1-A539, M1-C538, M1-P537, M1-E536, M1-V535, M1-L534, M1-L533,M1-D532, M1-F531, M1-N530, M1-A529, M1-H528, M1-E527, M1-L526, M1-E525,M1-E524, M1-I523, M1-S522, M1-I521, M1-G520, M1-S519, M1-K518, M1-R517,M1-Q516, M1-M515, M1-S514, M1-C513, M1-L512, M1-R511, M1-S510, M1-H509,M1-A508, M1-Y507, M1-Q506, M1-L505, M1-Y504, M1-P503, M1-G502, M1-T501,M1-D500, M1-G499, M1-E498, M1-F497, M1-S496, M1-T495, M1-M494, M1-R493,M1-D492, M1-W491, M1-K490, M1-F489, M1-E488, M1-Y487, M1-N486, M1-H485,M1-I484, M1-R483, M1-K482, M1-S481, M1-Q480, M1-M479, M1-D478, M1-Q477,M1-I476, M1-M475, M1-V474, M1-A473, M1-S472, M1-I471, M1-G470, M1-I469,M1-L468, M1-D467, M1-A466, M1-I465, M1-K464, M1-D463, M1-P462, M1-D461,M1-E460, M1-I459, M1-Q458, M1-A457, M1-Y456, M1-K455, M1-E454, M1-E453,M1-N452, M1-K451, M1-Q450, M1-M449, M1-V448, M1-E447, M1-H446, M1-M445,M1-K444, M1-E443, M1-K442, M1-T441, M1-E440, M1-Q439, M1-L438, M1-I437,M1-N436, M1-D435, M1-L434, M1-F433, M1-V432, M1-V431, M1-T430, M1-G429,M1-K428, M1-R427, M1-T426, M1-S425, M1-M424, M1-G423, M1-Q422, M1-V421,M1-M420, M1-G419, M1-F418, M1-N417, M1-V416, M1-H415, M1-E414, M1-L413,M1-N412, M1-H411, M1-A410, M1-W409, M1-E408, M1-F407, M1-G406, M1-M405,M1-Q404, M1-K403, M1-L402, M1-I401, M1-E400, M1-F399, M1-F398, M1-Q397,M1-A396, M1-C395, M1-H394, M1-L393, M1-D392, M1-Q391, M1-Q390, M1-A389,M1-A388, M1-I387, M1-V386, M1-Y385, M1-I384, M1-M383, M1-K382, M1-D381,M1-F380, M1-K379, M1-Y378, M1-T377, M1-E376, M1-Y375, M1-R374, M1-K373,M1-I372, M1-A371, M1-E370, M1-G369, M1-V368, M1-D367, M1-R366, M1-T365,M1-L364, M1-Y363, M1-L362, M1-S361, M1-T360, M1-G359, M1-D358, M1-S357,M1-K356, M1-E355, M1-V354, M1-L353, M1-A352, M1-K351, M1-G350, M1-L349,M1-K348, M1-K347, M1-N346, M1-F345, M1-K344, M1-T343, M1-L342, M1-D341,M1-I340, M1-L339, M1-K338, M1-A337, M1-G336, M1-R335, M1-D334, M1-I333,M1-D332, M1-I331, M1-L330, M1-G329, M1-K328, M1-D327, M1-E326, M1-F325,M1-L324, M1-K323, M1-T322, M1-A321, M1-E320, M1-K319, M1-M318, M1-K317,M1-E316, M1-Q315, M1-P314, M1-V313, M1-Q312, M1-S311, M1-E310, M1-G309,M1-S308, M1-Y307, M1-V306, M1-D305, M1-Y304, M1-K303, M1-I302, M1-N301,M1-L300, M1-R299, M1-G298, M1-Y297, M1-T296, M1-D295, M1-V294, M1-Y293,M1-K292, M1-E291, M1-I290, M1-S289, M1-L288, M1-D287, M1-R286, M1-F285,M1-R284, M1-A283, M1-W282, M1-I281, M1-K280, M1-L279, M1-A278, M1-S277,M1-E276, M1-D275, M1-G274, M1-D273, M1-E272, M1-M271, M1-R270, M1-R269,M1-F268, M1-F267, M1-R266, M1-R265, M1-A264, M1-E263, M1-E262, M1-N261,M1-T260, M1-S259, M1-S258, M1-Q257, M1-I256, M1-K255, M1-K254, M1-E253,M1-D252, M1-Q251, M1-E250, M1-S249, M1-A248, M1-D247, M1-I246, M1-T245,M1-E244, M1-A243, M1-P242, M1-T241, M1-E240, M1-G239, M1-T238, M1-A237,M1-E236, M1-S235, M1-T234, M1-E233, M1-K232, M1-T231, M1-V230, M1-D229,M1-Q228, M1-N227, M1-I226, M1-K225, M1-V224, M1-Y223, M1-V222, M1-E221,M1-F220, M1-L219, M1-H218, M1-N217, M1-I216, M1-P215, M1-D214, M1-S213,M1-A212, M1-L211, M1-K210, M1-S209, M1-E208, M1-D207, M1-G206, M1-Y205,M1-R204, M1-E203, M1-F202, M1-G201, M1-V200, M1-A199, M1-L198, M1-L197,M1-G196, M1-F195, M1-Q194, M1-K193, M1-G192, M1-W191, M1-D190, M1-G189,M1-L188, M1-Y187, M1-N186, M1-I185, M1-R184, M1-T183, M1-V182, M1-D181,M1-W180, M1-G179, M1-V178, M1-K177, M1-E176, M1-Y175, M1-L174, M1-N173,M1-S172, M1-I171, M1-F170, M1-G169, M1-G168, M1-I167, M1-I166, M1-T165,M1-S164, M1-R163, M1-L162, M1-H161, M1-G160, M1-A159, M1-H158, M1-F157,M1-P156, M1-K155, M1-A154, M1-I153, M1-N152, M1-P151, M1-S150, M1-S149,M1-F148, M1-E147, M1-V146, M1-I145, M1-A144, M1-K143, M1-K142, M1-G141,M1-V140, M1-G139, M1-L138, M1-P137, M1-L136, M1-Y135, M1-G134, M1-Y133,M1-D132, M1-S131, M1-K130, M1-R129, M1-K128, M1-L127, M1-V126, M1-D125,M1-E124, M1-I123, M1-V122, M1-L121, M1-N120, M1-Y119, M1-L118, M1-L117,M1-T116, M1-K115, M1-A114, M1-F113, M1-Y112, M1-F111, M1-Q110, M1-L109,M1-F108, M1-V107, M1-G106, M1-Q105, M1-P104, M1-K103, M1-I102, M1-E11,M1-S100, M1-I99, M1-F98, M1-K97, M1-G96, M1-K95, M1-N94, M1-F93, M1-N92,M1-E91, M1-A90, M1-W89, M1-E88, M1-K87, M1-S86, M1-K85, M1-E84, M1-N83,M1-P82, M1-N81, M1-I80, M1-G79, M1-K78, M1-L77, M1-R76, M1-L75, M1-K74,M1-P73, M1-I72, M1-P71, M1-V70, M1-I69, M1-I68, M1-D67, M1-G66, M1-Q65,M1-D64, M1-L63, M1-V62, M1-K61, M1-P60, M1-T59, M1-D58, M1-L57, M1-A56,M1-Q55, M1-I54, M1-I53, M1-I52, M1-S51, M1-K50, M1-D49, M1-V48, M1-S47,M1-S46, M1-I45, M1-R44, M1-H43, M1-L42, M1-E41, M1-E40, M1-A39, M1-I38,M1-Y37, M1-N36, M1-R35, M1-F34, M1-V33, M1-D32, M1-V31, M1-V30, M1-N29,M1-Y28, M1-Q27, M1-P26, M1-H25, M1-T24, M1-G23, M1-E22, M1-I21, M1-A20,M1-A19, M1-P18, M1-Q17, M1-S16, M1-L15, M1-G14, M1-L13, M1-Q12, M1-K11,M1-L10, M1-S9, M1-D8, and/or M1-S7 of SEQ ID NO:13. Polynucleotidesequences encoding these polypeptides are also provided. The presentinvention also encompasses the use of these C-terminal CaYDR341cdeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polynucleotide sequence provided in SEQ ID NO:2, andin particular to the coding region of the polynucleotide sequenceprovided in SEQ ID NO:2. Preferably such polynucleotides encodepolypeptides that have biological activity, particularly arginyl-tRNAsynthetase activity.

Most preferred are polynucleotides that share at least about 96.8%identity with the polynucleotide sequence provided in SEQ ID NO:2.

The present invention also encompasses polypeptides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polypeptide sequence provided in SEQ ID NO:12.

Most preferred are polypeptides that share at least about 96.8% identitywith the polypeptide sequence provided in SEQ ID NO:12.

The present invention is also directed to a homology model detailing thethree-dimensional structure of the CaYDR341c polypeptide (SEQ ID NO:13)of the present invention.

Protein threading and molecular modeling of CaYDR341c suggest thatCaYDR341c has a three dimensional fold similar to that of thearginyl-tRNA synthetase (EC number 6.1.1.-) from yeast (Delagoutte et.al., 2000), Protein Data Bank (PDB, Bernstein et. al., 1977 & Berman et.al., 2000) (Protein Data Bank entry 1F7U; Genbank Accession No.gil14719542; SEQ ID NO:252). Based on sequence, structure, motifs andknown RNA binding signature sequences, CaYDR341c contains a novel RNAsynthetase class 1 domain.

The polypeptide CaYDR341c contains several distinct structural domainsincluding the arginyl-tRNA synthetase catalytic domain which containsthe active site.

The three dimensional crystallographic structure for severalarginyl-tRNA synthetases have been reported and are deposited into theProtein Data Bank (Delagoutte et. al., 2000, Bernstein et. al., 1977,Berman et. al., 2000). The structure (Protein Data Bank, PDB entry 1F7u)of the arginyl-tRNA synthetase from yeast (Saccharomyces cerevisiae) issimilar to the other aminoacyl-tRNA synthetases (EC 6.1.1.-). Theyconstitute a family of RNA-binding proteins that are responsible for thecorrect translation of the genetic code by linking the 3′ end of thecorrect tRNA. In most organisms there are 20 aminoacyl-tRNA synthetases,each one responsible for its cognate tRNA(s). The structure of the yeast(Saccharomyces cerevisiae) arginyl-tRNA synthetase has the catalyticdomain at the core to which four structurally defined domains areappended. Domains attached to the N-terminus and C-terminus are definedas Add1 and Add2, respectively. The other two domains (Ins1 and Ins2)are inserted into regions of the catalytic core.

The arginyl-tRNA synthetase enzyme is a ternery complex with tRNA^(arg)and forms an extensive interface with a large burried surface areadescribed in detail by Delagoutte et. al., 2000. The interactionsbetween the enzyme and RNA include (I) exposed aromatic and alaphaticinteractions that are involved in van der Waals and hydrophobicintereactions; (II) positively charged residues that interact with thesugar phosphate backbone; (III) polar side chains for hydrogen bondsdirectly with nucleic acids or water-mediated hydrogen bonds. Therecognition surface is marked by a series of amino acids that areconserved in all known arginyl-tRNA synthetases (e.g. Y491, A495, R501,Y565, and M607). In addition a structural motif known as the Ω loop,which contains residues 480-485, forms a critical protruding loop thathas a dual functional role by forming part of a binding pocket andstabilizing the tRNA conformation by correct positioning of the RNAanticodon. A second structural feature that several aminoacyl-tRNAsynthetases share is located in the N-terminal region and is atetrapeptide sequence ‘HIGH’ (SEQ ID NO:255). The ‘HIGH’ (SEQ ID NO:255)region has been shown (Brick et al. 1988) to be part of the adenylatebinding site. The ‘HIGH’ (SEQ ID NO:255) signature has been found in theaminoacyl-tRNA synthetases specific for arginine, cysteine, glutamicacid, glutamine, isoleucine, leucine, methionine, tyrosine, tryptophan,and valine. These aminoacyl-tRNA synthetases are referred to as class-Isynthetases (Delarue and Moras, 1993; Schimmel, 1991) and seem to sharethe same core domain tertiary structure based on a Rossmann fold.

This structure-based information and sequence information from novelgenes can be used to identify other protein family members that sharethis same fold.

The three dimensional model of the CaYDR341c polypeptide provides for aspecific description of the catalytic core and functional sites in thearginyl-tRNA synthetase, CaYDR341c polypeptide.

The catalytic core and functional sites are defined by atomiccoordinates (Table 9). Based on this data, the inventors have ascribedthe CaYDR341c polypeptide as having arginyl-tRNA synthetase activity(s)and cellular and systemic regulatory function(s).

The invention also relates to in silico screening methods including insilico docking and methods of structure based drug design which utilizethe three dimensional coordinates of CaYDR341c (Table 9). Also providedare methods of identifying modulators of CaYDR341c that includemodulator building or searching utilizing computer programs andalgorithms. In an embodiment of the invention a method is provided fordesigning potential modulators of CaYDR341c comprising any combinationof steps which utilize said three dimensional structure to design orselect potential modulators.

Homology models are useful when there is no experimental informationavailable on the protein of interest. A three dimensional model can beconstructed on the basis of the known structure of a homologous protein(Greer et. al., 1991, Lesk, et. al., 1992, Levitt, 1992, Cardozo, et.al., 1995, Sali, et. al., 1995).

Those of skill in the art will understand that a homology model isconstructed on the basis of first identifying a template, or, protein ofknown structure which is similar to the protein without known structure.This can be accomplished by pairwise alignment of sequences using suchprograms as FASTA (Pearson, et. al. 1990) and BLAST (Altschul, et. al.,1990). In cases where sequence similarity is high (greater than 30%),these pairwise comparison methods may be adequate. Likewise, multiplesequence alignments or profile-based methods can be used to align aquery sequence to an alignment of multiple (structurally andbiochemically) related proteins. When the sequence similarity is low,more advanced techniques are used such as fold recognition (proteinthreading; Hendlich, et. al., 1990, Koppensteiner et. Al. 2000, Sippl &Weitckus 1992, Sippl 1993), where the compatibility of a particularsequence with the three dimensional fold of a potential template proteinis gauged on the basis of a knowledge-based potential. Following theinitial sequence alignment, the query template can be optimally alignedby manual manipulation or by incorporation of other features (motifs,secondary structure predictions, and allowed sequence conservation).Next, structurally conserved regions can be identified and are used toconstruct the core secondary structure (Levitt, 1992, Sali, et. al.,1995) elements in the three dimensional model. Variable regions, called“unconserved regions” and loops can be added using knowledge-basedtechniques. The complete model with variable regions and loops can berefined performing forcefield calculations (Sali, et. al., 1995,Cardozo, et. al., 1995).

For CaYDR341c a pairwise alignment generated by FASTA was used to alignthe sequence of CaYDR341c with the sequence the arginyl-tRNA synthetase,EC number 6.1.1.- from yeast, Saccharomyces cerevisiae (Delagoutte et.al., 2000), (Protein Data Bank code 1F7U). The alignment of CaYDR341cwith PDB entry 1F7U chain A is set forth in FIG. 29. In this invention,the homology model of CaYDR341c was derived from the sequence alignmentset forth in FIG. 29. An overall atomic model including plausiblesidechain orientations was generated using the program LOOK (Levitt,1992). The three dimensional model for CaYDR341c is defined by the setof structure coordinates as set forth in Table 9 and is shown in FIG. 31rendered by backbone secondary structures.

In order to recognize errors in three-dimensional structures, knowledgebased mean fields can be used to judge the quality of protein folds(Sippl 1993). The methods can be used to recognize misfolded structuresas well as faulty parts of structural models. The technique generates anenergy graph where the energy distribution for a given protein fold isdisplayed on the y-axis and residue position in the protein fold isdisplayed on the x-axis. The knowledge based mean fields compose a forcefield derived from a set of globular protein structures taken as asubset from the Protein Data Bank (Bernstein et. al. 1977). To analyzethe quality of a model, the energy distribution is plotted and comparedto the energy distribution of the template from which the model wasgenerated. FIG. 31 shows the energy graph for the CaYDR341c model(dotted line) and the template (arginyl-tRNA synthetase) from which themodel was generated. The model has virtually an identical energy plotwhen compared to arginyl-tRNA synthetase template demonstrating thatCaYDR341c has similar structural characteristics and suggest the overallthree-dimensional fold is “native-like”. This graph supports the motifand sequence alignments in confirming that the three dimensionalstructure coordinates of CaYDR341c are an accurate and usefulrepresentation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model.

Those of skill in the art will understand that a set of structurecoordinates for a protein is a relative set of points that define ashape in three dimensions. Thus, it is possible that an entirelydifferent set of coordinates could define a similar or identical shape.Moreover, slight variations in the individual coordinates, as emanatefrom generation of similar homology models using different alignmenttemplates (i.e., other than the structure coordinates of 1F7u), and/orusing different methods in generating the homology model, will haveminor effects on the overall shape. Variations in coordinates may alsobe generated because of mathematical manipulations of the structurecoordinates. For example, the structure coordinates set forth in Table 9could be manipulated by fractionalization of the structure coordinates;integer additions or subtractions to sets of the structure coordinates,inversion of the structure coordinates or any combination of the above.

Various computational analyses are therefore necessary to determinewhether a molecule, or a portion thereof, is sufficiently similar to allor parts of CaYDR341c described above as to be considered the same. Suchanalyses may be carried out in current software applications, such asINSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as describedin the User's Guide, online or software applications available in theSYBYL software suite (Tripos Inc., St. Louis, Mo.).

Using the superimposition tool in the program INSIGHTII comparisons canbe made between different structures and different conformations of thesame structure. The procedure used in INSIGHTII to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. Since atom equivalency within INSIGHTII isdefined by user input, for the purpose of this invention, equivalentatoms are defined as protein backbone atoms (N, Cα, C and O) for allconserved residues between the two structures being compared. Also, onlyrigid fitting operations are considered. When a rigid fitting method isused, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atom is anabsolute minimum. This number, given in angstroms, is reported byINSIGHTII.

For the purpose of this invention, any homology model of a CaYDR341cthat has a root mean square deviation of conserved residue backboneatoms (N, Cα, C, O) of less than about 2.0 A when superimposed on therelevant backbone atoms described by structure coordinates listed inTable 9 are considered identical. More preferably, the root mean squaredeviation is less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, or 0.1 Angstroms.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a protein from the relevant portion of thebackbone of CaYDR341c as defined by the structure coordinates describedherein.

This invention as embodied by the three-dimensional model enables thestructure-based design of modulators of the biological function ofCaYDR341c, as well as mutants with altered biological function and/orspecificity.

The sequence alignment (FIG. 29) used as a template for creating thethree-dimensional model of CaYDR341c arginyl-tRNA synthetase domainshows 67% sequence identity between catalytic domain of CaYDR341c andyeast arginyl-tRNA synthetase, PDB code 1F7U. For the arginyl-tRNAsynthetases there are at least two functional regions that are critical.In the N-terminal region of the enzyme the adenylate binding site hasbeen shown to be highly conserved. The motifs and structure of thisregion are similar for class I synthetases aminoacyl-tRNA synthetasesspecific for arginine, cysteine, glutamic acid, glutamine, isoleucine,leucine, methionine, tyrosine, tryptophan, and valine (Brick et al.1988). FIG. 29 shows this region highlighted by (*) and corresponds toH158-H161 in the three dimensional model for CaYDR341c (Table 9). Theadenylate binding site, including the canonical “HIGH” (SEQ ID NO:255)motif, and surrounding sequence is completely conserved at the sequenceand structure level. The Ω loop in aminoacyl-tRNA synthetases forms acritical protruding structure that functions by forming part of abinding pocket for the tRNA molecule and stabilizing the tRNAconformation by correct positioning of the tRNA anticodon. FIG. 29 showsthat the Ω loop in CaYDR341c is completely conserved (denoted by “+”)both in sequence and structurally.

The conservation of the amino acids in both of these functional sites,and the overall 67% sequence identity, emphasizes the significance ofthe CaYDR341c three-dimensional model. The conserved residues arelocated in the functional sites at the tRNA interface presenting a wellstructured catalytic domain. These functional site residues playcritical roles in the mechanism of catalysis, substrate specificity andtRNA binding.

The structure coordinates of a CaYDR341c homology model, and portionsthereof, are stored in a machine-readable storage medium. Such data maybe used for a variety of purposes, such as drug discovery and targetprioritization and validation.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table 9.

For the first time, the present invention permits the use, throughhomology modeling based upon the sequence of CaYDR341c (FIG. 12) ofstructure-based or rational drug design techniques to design, select,and synthesizes chemical entities that are capable of modulating thebiological function of CaYDR341c. Comparison of the CaYDR341c homologymodel with the structures of other the arginyl-tRNA synthetases enablethe use of rational or structure based drug design methods to design,select or synthesize specific chemical modulators of CaYDR341c.

Accordingly, the present invention is also directed to the entiresequence in FIG. 12, or any portion thereof, for the purpose ofgenerating a homology model for the purpose of three dimensionalstructure-based drug designs.

The present invention also encompasses mutants or homologues of thesequence in FIG. 12, or any portion thereof. In a preferred embodiment,the mutants or homologues have at least 25% identity, more preferably50% identity, more preferably 75% identity, and most preferably 90%identity to the amino acid residues in FIG. 12.

The three-dimensional model structure of the CaYDR341c will also providemethods for identifying modulators of biological function. Variousmethods or combination thereof can be used to identify these compounds.

Structure coordinates of the active site region defined above can alsobe used to identify structural and chemical features. Identifiedstructural or chemical features can then be employed to design or selectcompounds as potential CaYDR341c modulators. By structural and chemicalfeatures it is meant to include, but is not limited to, van der Waalsinteractions, hydrogen bonding interactions, charge interaction,hydrophobic interactions, and dipole interaction. Alternatively, or inconjunction, the three-dimensional structural model can be employed todesign or select compounds as potential CaYDR341c modulators. Compoundsidentified as potential CaYDR341c modulators can then be synthesized andscreened in an assay characterized by binding of a test compound to theCaYDR341c, or in characterizing CaYDR341c deactivation in the presenceof a small molecule. Examples of assays useful in screening of potentialCaYDR341c modulators include, but are not limited to, screening insilico, in vitro assays and high throughput assays. Finally, thesemethods may also involve modifying or replacing one or more amino acidsfrom CaYDR341c according to Table 9.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number of computer modeling systems are available inwhich the sequence of the CaYDR341c and the CaYDR341c structure (i.e.,atomic coordinates of CaYDR341c and/or the atomic coordinates of theactive site region as provided in Table 9) can be input. The computersystem then generates the structural details of one or more theseregions in which a potential CaYDR341c modulator binds so thatcomplementary structural details of the potential modulators can bedetermined. Design in these modeling systems is generally based upon thecompound being capable of physically and structurally associating withCaYDR341c. In addition, the compound must be able to assume aconformation that allows it to associate with CaYDR341c. Some modelingsystems estimate the potential inhibitory or binding effect of apotential CaYDR341c modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are well known. Often thesemethods begin by visual inspection of the binding site on the computerscreen. Selected fragments or chemical entities are then positioned inone or more positions and orientations within the active site region inCaYDR341c. Molecular docking is accomplished using software such asINSIGHTII, ICM (Molsoft LLC, La Jolla, Calif.), and SYBYL, following byenergy minimization and molecular dynamics with standard molecularmechanic forcefields such as CHARMM and MMFF. Examples of computerprograms which assist in the selection of chemical fragment or chemicalentities useful in the present invention include, but are not limitedto, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntzet. al. 1982).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992), LeapFrog (Tripos Associates, St. Louis Mo.) and DOCK (Kuntzet. al., 1982). Programs such as DOCK (Kuntz et. al. 1982) can be usedwith the atomic coordinates from the homology model to identifypotential ligands from databases or virtual databases which potentiallybind the in the active site region, and which may therefore be suitablecandidates for synthesis and testing. The computer programs may utilizea combination of the following steps:

1) Selection of fragments or chemical entities from a database and thenpositioning the chemical entity in one or more orientations within theCaYDR341c catalytic domain defined by Table 9

2) Characterization of the structural and chemical features of thechemical entity and active site including van der Waals interactions,hydrogen bonding interactions, charge interaction, hydrophobic bondinginteraction, and dipole interactions

3) Search databases for molecular fragments which can be joined to orreplace the docked chemical entity and spatially fit into regionsdefined by the said CaYDR341c catalytic domain or catalytic domainfunctional sites

4) Evaluate the docked chemical entity and fragments using a combinationof scoring schemes which account for van der Waals interactions,hydrogen bonding interactions, charge interaction, hydrophobicinteractions

Databases that may be used include ACD (Molecular Designs Limited),Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute),Maybridge (Maybridge Chemical Company Ltd), CCDC (CambridgeCrystallographic Data Center), CAST (Chemical Abstract Service), Derwent(Derwent Information Limited).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and CaYDR341c can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm1992) as well as 3D Database systems (Martin 1992).

Additionally, the three-dimensional homology model of CaYDR341c will aidin the design of mutants with altered biological activity. Site directedmutagenesis can be used to generate proteins with similar or varyingdegrees of biological activity compared to native CaYDR341c. Thisinvention also relates to the generation of mutants or homologs ofCaYDR341c. It is clear that molecular modeling using the threedimensional structure coordinates set forth in Table 9 and visualizationof the CaYDR341c model, FIG. 31 can be utilized to design homologs ormutant polypeptides of CaYDR341c that have similar or altered biologicalactivities, function or reactivities.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:2 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 1852 ofSEQ ID NO:2, b is an integer between 15 to 1866, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:2,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:3

The polynucleotide sequence (SEQ ID NO:3) and deduced amino acidsequence (SEQ ID NO:14) of the novel fungal essential gene, CaYLR022c(also referred to as FCG7), of the present invention. The CaYLR022cpolypeptide (SEQ ID NO:14) is encoded by nucleotides 1 to 765 of SEQ IDNO:3 and has a predicted molecular weight of 29.2 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYLR022c. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 765 of SEQ ID NO:3, and the polypeptide corresponding to aminoacids 2 thru 255 of SEQ ID NO:15. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYLR022c polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYLR022c deletionpolypeptides are encompassed by the present invention: M1-E255, A2-E255,V3-E255, I4-E255, N5-E255, Q6-E255, P7-E255, N8-E255, S9-E255, Q10-E255,I11-E255, R12-E255, L13-E255, T14-E255, N15-E255, V16-E255, S17-E255,L18-E255, V19-E255, R20-E255, M21-E255, K22-E255, K23-E255, G24-E255,K25-E255, K26-E255, R27-E255, F28-E255, E29-E255, I30-E255, A31-E255,C32-E255, Y33-E255, Q34-E255, N35-E255, K36-E255, V37-E255, Q38-E255,D39-E255, W40-E255, R41-E255, L42-E255, K43-E255, V44-E255, E45-E255,K46-E255, D47-E255, I48-E255, D49-E255, E50-E255, V51-E255, L52-E255,Q53-E255, I54-E255, P55-E255, Q56-E255, V57-E255, F58-E255, I59-E255,N60-E255, V61-E255, S62-E255, K63-E255, G64-E255, Q65-E255, V66-E255,A67-E255, N68-E255, N69-E255, D70-E255, D71-E255, L72-E255, Q73-E255,K74-E255, C75-E255, F76-E255, G77-E255, T78-E255, T79-E255, N80-E255,Q81-E255, D82-E255, E83-E255, I84-E255, I85-E255, A86-E255, E87-E255,I88-E255, L89-E255, N90-E255, K91-E255, G92-E255, E93-E255, I94-E255,Q95-E255, L96-E255, N97-E255, E98-E255, K99-E255, E100-E255, R101-E255,N102-E255, A103-E255, N104-E255, L105-E255, Q106-E255, Q107-E255,K108-E255, Q109-E255, N110-E255, E111-E255, F112-E255, L113-E255,N114-E255, I115-E255, I116-E255, S117-E255, T118-E255, K119-E255,C120-E255, I121-E255, N122-E255, P123-E255, R124-E255, S125-E255,K126-E255, K127-E255, R128-E255, Y129-E255, P130-E255, P131-E255,S132-E255, M133-E255, I134-E255, E135-E255, K136-E255, V137-E255,L138-E255, N139-E255, E140-E255, V141-E255, K142-E255, F143-E255,H144-E255, L145-E255, N146-E255, P147-E255, T148-E255, K149-E255,P150-E255, T151-E255, K152-E255, I153-E255, Q154-E255, A155-E255,L156-E255, D157-E255, A158-E255, I159-E255, K160-E255, L161-E255,L162-E255, V163-E255, E164-E255, K165-E255, Q166-E255, I167-E255,I168-E255, P169-E255, I170-E255, A171-E255, R172-E255, A173-E255,Q174-E255, M175-E255, K176-E255, V177-E255, R178-E255, I179-E255,T180-E255, L181-E255, S182-E255, K183-E255, K184-E255, A185-E255,Y186-E255, L187-E255, K188-E255, T189-E255, F190-E255, Q191-E255,D192-E255, E193-E255, I194-E255, K195-E255, P196-E255, V197-E255,I98-E255, D199-E255, Q200-E255, I201-E255, V202-E255, E203-E255,E204-E255, D205-E255, N206-E255, N207-E255, G208-E255, K209-E255,Q210-E255, Y211-E255, E212-E255, I213-E255, V214-E255, G215-E255,I216-E255, I217-E255, D218-E255, P219-E255, I220-E255, N221-E255,Y222-E255, R223-E255, V224-E255, L225-E255, V226-E255, T227-E255,L228-E255, I229-E255, E230-E255, N231-E255, T232-E255, D233-E255,G234-E255, S235-E255, N236-E255, K237-E255, V238-E255, A239-E255,K240-E255, G241-E255, E242-E255, G243-E255, S244-E255, I245-E255,E246-E255, V247-E255, L248-E255, and/or D249-E255 of SEQ ID NO:14.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalCaYLR022c deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

In preferred embodiments, the following C-terminal CaYLR022c deletionpolypeptides are encompassed by the present invention: M1-E255, M1-K254,M1-I253, M1-A252, M1-S251, M1-M250, M1-D249, M1-L248, M1-V247, M1-E246,M1-I245, M1-S244, M1-G243, M1-E242, M1-G241, M1-K240, M1-A239, M1-V238,M1-K237, M1-N236, M1-S235, M1-G234, M1-D233, M1-T232, M1-N231, M1-E230,M1-I229, M1-L228, M1-T227, M1-V226, M1-L225, M1-V224, M1-R223, M1-Y222,M1-N221, M1-I220, M1-P219, M1-D218, M1-I217, M1-I216, M1-G215, M1-V214,M1-I213, M1-E212, M1-Y211, M1-Q210, M1-K209, M1-G208, M1-N207, M1-N206,M1-D205, M1-E204, M1-E203, M1-V202, M1-I201, M1-Q200, M1-D199, M1-I198,M1-V197, M1-P196, M1-K195, M1-I194, M1-E193, M1-D192, M1-Q191, M1-F190,M1-T189, M1-K188, M1-L187, M1-Y186, M1-A185, M1-K184, M1-K183, M1-S182,M1-L181, M1-T180, M1-I179, M1-R178, M1-V177, M1-K176, M1-M175, M1-Q174,M1-A173, M1-R172, M1-A171, M1-I170, M1-P169, M1-I168, M1-I167, M1-Q166,M1-K165, M1-E164, M1-V163, M1-L162, M1-L161, M1-K160, M1-I159, M1-A158,M1-D157, M1-L156, M1-A155, M1-Q154, M1-I153, M1-K152, M1-T151, M1-P150,M1-K149, M1-T148, M1-P147, M1-N146, M1-L145, M1-H144, M1-F143, M1-K142,M1-V141, M1-E140, M1-N139, M1-L138, M1-V137, M1-K136, M1-E135, M1-I134,M1-M133, M1-S132, M1-P131, M1-P130, M1-Y129, M1-R128, M1-K127, M1-K126,M1-S125, M1-R124, M1-P123, M1-N122, M1-I121, M1-C120, M1-K119, M1-T118,M1-S117, M1-I116, M1-I115, M1-N114, M1-L113, M1-F112, M1-E111, M1-N110,M1-Q109, M1-K108, M1-Q107, M1-Q106, M1-L105, M1-N104, M1-A103, M1-N102,M1-R10, M1-E10, M1-K99, M1-E98, M1-N97, M1-L96, M1-Q95, M1-I94, M1-E93,M1-G92, M1-K91, M1-N90, M1-L89, M1-I88, M1-E87, M1-A86, M1-I85, M1-I84,M1-E83, M1-D82, M1-Q81, M1-N80, M1-T79, M1-T78, M1-G77, M1-F76, M1-C75,M1-K74, M1-Q73, M1-L72, M1-D71, M1-D70, M1-N69, M1-N68, M1-A67, M1-V66,M1-Q65, M1-G64, M1-K63, M1-S62, M1-V61, M1-N60, M1-I59, M1-F58, M1-V57,M1-Q56, M1-P55, M1-I54, M1-Q53, M1-L52, M1-V51, M1-E50, M1-D49, M1-I48,M1-D47, M1-K46, M1-E45, M1-V44, M1-K43, M1-L42, M1-R41, M1-W40, M1-D39,M1-Q38, M1-V37, M1-K36, M1-N35, M1-Q34, M1-Y33, M1-C32, M1-A31, M1-I30,M1-E29, M1-F28, M1-R27, M1-K26, M1-K25, M1-G24, M1-K23, M1-K22, M1-M21,M1-R20, M1-V19, M1-L18, M1-S17, M1-V16, M1-N15, M1-T14, M1-L13, M1-R12,M1-I11, M1-Q10, M1-S9, M1-N8, and/or M1-P7 of SEQ ID NO:14.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these C-terminalCaYLR022c deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polynucleotide sequence provided in SEQ ID NO:3, andin particular to the coding region of the polynucleotide sequenceprovided in SEQ ID NO:3. Preferably such polynucleotides encodepolypeptides that have biological activity.

The present invention also encompasses polypeptides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polypeptide sequence provided in SEQ ID NO:13.

Most preferred are polypeptides that share at least about 99.7% identitywith the polypeptide sequence provided in SEQ ID NO:13.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:3 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 751 ofSEQ ID NO:3, b is an integer between 15 to 765, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:3,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:4

The polynucleotide sequence (SEQ ID NO:4) and deduced amino acidsequence (SEQ ID NO:15) of the novel fungal essential gene, CaYOL077c(also referred to as FCG8), of the present invention. The CaYOL077cpolypeptide (SEQ ID NO:15) is encoded by nucleotides 1 to 876 of SEQ IDNO:4 and has a predicted molecular weight of 34.0 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYOL077c. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 876 of SEQ ID NO:4, and the polypeptide corresponding to aminoacids 2 thru 292 of SEQ ID NO:16. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYOL077c polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYOL077c deletionpolypeptides are encompassed by the present invention: M1-K292, S2-K292,A3-K292, I4-K292, Y5-K292, K6-K292, A7-K292, L8-K292, Q9-K292, S10-K292,K11-K292, S12-K292, S13-K292, K14-K292, E15-K292, T16-K292, S17-K292,E18-K292, K19-K292, T20-K292, K21-K292, H22-K292, I23-K292, N24-K292,R25-K292, Q26-K292, R27-K292, L28-K292, L29-K292, V30-K292, I31-K292,S32-K292, S33-K292, R34-K292, G35-K292, I36-K292, T37-K292, Y38-K292,R39-K292, H40-K292, R41-K292, H42-K292, L43-K292, I44-K292, Q45-K292,D46-K292, L47-K292, L48-K292, A49-K292, L50-K292, L51-K292, P52-K292,H53-K292, A54-K292, R55-K292, K56-K292, E57-K292, P58-K292, K59-K292,F60-K292, D61-K292, S62-K292, K63-K292, K64-K292, N65-K292, L66-K292,H67-K292, Q68-K292, L69-K292, N70-K292, E71-K292, V72-K292, A73-K292,E74-K292, L75-K292, Y76-K292, N77-K292, C78-K292, N79-K292, N80-K292,I81-K292, F82-K292, F83-K292, F84-K292, E85-K292, C86-K292, R87-K292,K88-K292, H89-K292, Q90-K292, D91-K292, L92-K292, Y93-K292, L94-K292,W95-K292, I96-K292, S97-K292, K98-K292, P99-K292, P100-K292, N101-K292,G102-K292, P103-K292, T104-K292, L105-K292, K106-K292, F107-K292,H108-K292, I109-K292, Q110-K292, N111-K292, L112-K292, H113-K292,T114-K292, L115-K292, D116-K292, E117-K292, L118-K292, N119-K292,F120-K292, T121-K292, G122-K292, N123-K292, C124-K292, L125-K292,K126-K292, G127-K292, S128-K292, R129-K292, P130-K292, I131-K292,L132-K292, S133-K292, F134-K292, D135-K292, K136-K292, S137-K292,F138-K292, L139-K292, E140-K292, N141-K292, D142-K292, H143-K292,Y144-K292, K145-K292, L146-K292, L147-K292, K148-K292, E149-K292,M150-K292, F151-K292, L152-K292, Q153-K292, T154-K292, F155-K292,G156-K292, V157-K292, P158-K292, P159-K292, N160-K292, A161-K292,R162-K292, K163-K292, S164-K292, K165-K292, P166-K292, F167-K292,I168-K292, D169-K292, H170-K292, V171-K292, M172-K292, T173-K292,F174-K292, S175-K292, I176-K292, V177-K292, D178-K292, G179-K292,K180-K292, I181-K292, W182-K292, I183-K292, R184-K292, N185-K292,Y186-K292, Q187-K292, I188-K292, N189-K292, E190-K292, T191-K292,L192-K292, D193-K292, V194-K292, K195-K292, E196-K292, N197-K292,D198-K292, K199-K292, I200-K292, E201-K292, D202-K292, D203-K292,E204-K292, D205-K292, Y206-K292, D207-K292, V208-K292, D209-K292,Q210-K292, L211-K292, N212-K292, L213-K292, V214-K292, E215-K292,I216-K292, G217-K292, P218-K3 292, R219-K292, L220-K292, V221-K292,L222-K292, T223-K292, L224-K292, I225-K292, T226-K292, V227-K292,L228-K292, E229-K292, G230-K292, S231-K292, F232-K292, S233-K292,G234-K292, P235-K292, K236-K292, I237-K292, Y238-K292, E239-K292,N240-K292, K241-K292, Q242-K292, Y243-K292, V244-K292, S245-K292,P246-K292, N247-K292, F248-K292, V249-K292, R250-K292, A251-K292,Q252-K292, L253-K292, K254-K292, Q255-K292, Q256-K292, A257-K292,A258-K292, D259-K292, Q260-K292, A261-K292, K262-K292, S263-K292,R264-K292, S265-K292, Q266-K292, A267-K292, A268-K292, L269-K292,E270-K292, R271-K292, K272-K292, I273-K292, K274-K292, K275-K292,R276-K292, N277-K292, Q278-K292, V279-K292, L280-K292, K281-K292,A282-K292, D283-K292, P284-K292, L285-K292, and/or S286-K292 of SEQ IDNO: 15. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseN-terminal CaYOL077c deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYOL077c deletionpolypeptides are encompassed by the present invention: M1-K292, M1-F291,M1-L290, M1-A289, M1-D288, M1-N287, M1-S286, M1-L285, M1-P284, M1-D283,M1-A282, M1-K281, M1-L280, M1-V279, M1-Q278, M1-N277, M1-R276, M1-K275,M1-K274, M1-I273, M1-K272, M1-R271, M1-E270, M1-L269, M1-A268, M1-A267,M1-Q266, M1-S265, M1-R264, M1-S263, M1-K262, M1-A261, M1-Q260, M1-D259,M1-A258, M1-A257, M1-Q256, M1-Q255, M1-K254, M1-L253, M1-Q252, M1-A251,M1-R250, M1-V249, M1-F248, M1-N247, M1-P246, M1-S245, M1-V244, M1-Y243,M1-Q242, M1-K241, M1-N240, M1-E239, M1-Y238, M1-I237, M1-K236, M1-P235,M1-G234, M1-S233, M1-F232, M1-S231, M1-G230, M1-E229, M1-L228, M1-V227,M1-T226, M1-I225, M1-L224, M1-T223, M1-L222, M1-V221, M1-L220, M1-R219,M1-P218, M1-G217, M1-I216, M1-E215, M1-V214, M1-L213, M1-N212, M1-L211,M1-Q210, M1-D209, M1-V208, M1-D207, M1-Y206, M1-D205, M1-E204, M1-D203,M1-D202, M1-E201, M1-I200, M1-K199, M1-D198, M1-N197, M1-E196, M1-K195,M1-V194, M1-D193, M1-L192, M1-T191, M1-E190, M1-N189, M1-I188, M1-Q187,M1-Y186, M1-N185, M1-R184, M1-I183, M1-W182, M1-I181, M1-K180, M1-G179,M1-D178, M1-V177, M1-I176, M1-S175, M1-F174, M1-T173, M1-M172, M1-V171,M1-H170, M1-D169, M1-I168, M1-F167, M1-P166, M1-K165, M1-S164, M1-K163,M1-R162, M1-A161, M1-N160, M1-P159, M1-P158, M1-V157, M1-G156, M1-F155,M1-T154, M1-Q153, M1-L152, M1-F151, M1-M150, M1-E149, M1-K148, M1-L147,M1-L146, M1-K145, M1-Y144, M1-H143, M1-D142, M1-N141, M1-E140, M1-L139,M1-F138, M1-S137, M1-K136, M1-D135, M1-F134, M1-S133, M1-L132, M1-I131,M1-P130, M1-R129, M1-S128, M1-G127, M1-K126, M1-L125, M1-C124, M1-N123,M1-G122, M1-T121, M1-F120, M1-N119, M1-L118, M1-E117, M1-D116, M1-L115,M1-T114, M1-H113, M1-L112, M1-N111, M1-Q110, M1-I109, M1-H108, M1-F107,M1-K106, M1-L105, M1-T104, M1-P103, M1-G102, M1-N101, M1-P100, M1-P99,M1-K98, M1-S97, M1-I96, M1-W95, M1-L94, M1-Y93, M1-L92, M1-D91, M1-Q90,M1-H89, M1-K88, M1-R87, M1-C86, M1-E85, M1-F84, M1-F83, M1-F82, M1-I81,M1-N80, M1-N79, M1-C78, M1-N77, M1-Y76, M1-L75, M1-E74, M1-A73, M1-V72,M1-E71, M1-N70, M1-L69, M1-Q68, M1-H67, M1-L66, M1-N65, M1-K64, M1-K63,M1-S62, M1-D61, M1-F60, M1-K59, M1-P58, M1-E57, M1-K56, M1-R55, M1-A54,M1-H53, M1-P52, M1-L51, M1-L50, M1-A49, M1-L48, M1-L47, M1-D46, M1-Q45,M1-I44, M1-L43, M1-H42, M1-R41, M1-H40, M1-R39, M1-Y38, M1-T37, M1-I36,M1-G35, M1-R34, M1-S33, M1-S32, M1-I31, M1-V30, M1-L29, M1-L28, M1-R27,M1-Q26, M1-R25, M1-N24, M1-I23, M1-H22, M1-K21, M1-T20, M1-K19, M1-E18,M1-S17, M1-T16, M1-E15, M1-K14, M1-S13, M1-S12, M1-K11, M1-S10, M1-Q9,M1-L8, and/or M1-A7 of SEQ ID NO:15. Polynucleotide sequences encodingthese polypeptides are also provided. The present invention alsoencompasses the use of these C-terminal CaYOL077c deletion polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:4 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 862 ofSEQ ID NO:4, b is an integer between 15 to 876, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:4,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:5

The polynucleotide sequence (SEQ ID NO:5) and deduced amino acidsequence (SEQ ID NO:16) of the novel fungal essential gene, CaYNL132w(also referred to as FCG10), of the present invention. The CaYNL132wpolypeptide (SEQ ID NO:16) is encoded by nucleotides 1 to 3126 of SEQ IDNO:5 and has a predicted molecular weight of 117.3 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYNL132w. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 3126 of SEQ ID NO:5, and the polypeptide corresponding to aminoacids 2 thru 1042 of SEQ ID NO:17. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYNL132w polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYNL132w deletionpolypeptides are encompassed by the present invention: M1-K1042,G2-K1042, K3-K1042, K4-K1042, A5-K1042, I6-K1042, D7-K1042, A8-K1042,R9-K1042, I10-K1042, P11-K1042, A12-K1042, L13-K1042, I14-K1042,R15-K1042, N16-K1042, G17-K1042, V18-K1042, Q19-K1042, E20-K1042,K21-K1042, Q22-K1042, R23-K1042, S24-K1042, F25-K1042, F26-K1042,I27-K1042, I28-K1042, V29-K1042, G30-K1042, D31-K1042, K32-K1042,A33-K1042, R34-K1042, N35-K1042, Q36-K1042, L37-K1042, P38-K1042,N39-K1042, L40-K1042, H41-K. 1042, Y42-K1042, L43-K1042, M44-K1042,M45-K1042, S46-K1042, A47-K1042, D48-K1042, L49-K1042, K50-K1042,M51-K1042, N52-K1042, K53-K1042, S54-K1042, V55-K1042, L56-K1042,W57-K1042, A58-K1042, Y59-K1042, K60-K1042, K61-K1042, K62-K1042,L63-K1042, L64-K1042, G65-K1042, F66-K1042, T67-K1042, S68-K1042,H69-K1042, R70-K1042, Q71-K1042, K72-K1042, R73-K1042, E74-K1042,A75-K1042, K76-K1042, I77-K1042, K78-K1042, K79-K1042, D80-K1042,I81-K1042, K82-K1042, R83-K1042, G84-K1042, I85-K1042, R86-K1042,E87-K1042, V88-K1042, N89-K1042, E90-K1042, Q91-K1042, D92-K1042, 3P93-K1042, F94-K1042, E95-K1042, A96-K1042, F97-K1042, I98-K1042,S99-K1042, N100-K1042, Q101-K1042, H102-K1042, I103-K1042, R104-K1042,Y105-K1042, V106-K1042, Y107-K1042, Y108-K1042, K109-K1042, E110-K1042,T111-K1042, E112-K1042, K113-K1042, I114-K1042, L115-K1042, G116-K1042,N117-K1042, T118-K1042, Y19-K1042, G120-K1042, M121-K1042, C122-K1042,I123-K. 1042, L124-K1042, Q125-K1042, D126-K1042, F127-K1042,E128-K1042, A129-K1042, I130-K1042, T131-K1042, P132-K1042, N133-K1042,L134-K1042, L135-K1042, A136-K1042, R137-K1042, T138-K1042, I139-K1042,E140-K1042, T141-K1042, V142-K1042, E143-K1042, G144-K1042, G145-K1042,G146-K1042, L147-K1042, V148-K1042, V149-K1042, I150-K1042, L151-K1042,L152-K1042, K153-K1042, N154-K1042, M155-K1042, T156-K1042, S157-K1042,L158-K1042, K159-K1042, Q160-K1042, L161-K1042, Y162-K1042, T163-K1042,M164-K1042, S165-K1042, M166-K1042, D167-K1042, I168-K1042, H169-K1042,S170-K1042, R171-K1042, Y172-K1042, R173-K1042, T174-K1042, E175-K1042,A176-K1042, H177-K1042, D178-K1042, D179-K1042, V180-K1042, V181-K1042,A182-K1042, R183-K1042, F184-K1042, N185-K1042, E186-K1042, R187-K1042,F188-K1042, L189-K1042, L190-K1042, S191-K1042, L192-K1042, G193-K1042,S194-K1042, C195-K1042, E196-K1042, N197-K1042, C198-K1042, L199-K1042,V200-K1042, V201-K1042, D202-K1042, D203-K1042, E204-K1042, L205-K1042,N206-K1042, V207-K1042, L208-K1042, P209-K1042, I210-K1042, S211-K1042,G212-K1042, G213-K1042, K214-K1042, H215-K1042, V216-K1042, K217-K1042,P218-K1042, L219-K1042, P220-K1042, P221-K1042, K222-K1042, D223-K1042,D224-K1042, D225-K1042, E226-K1042, L227-K1042, T228-K1042, P229-K1042,N230-K1042, A231-K1042, K232-K1042, E233-K1042, L234-K1042, K235-K1042,E236-K1042, L237-K1042, K238-K1042, E239-K1042, S240-K1042, L241-K1042,A242-K1042, D243-K1042, V244-K1042, Q245-K1042, P246-K1042, A247-K1042,G248-K1042, S249-K1042, L250-K1042, V251-K1042, A252-K1042, L253-K1042,S254-K1042, K255-K1042, T256-K1042, I257-K1042, N258-K1042, Q259-K1042,A260-K1042, Q261-K1042, A262-K1042, I263-K1042, L264-K1042, T265-K1042,F266-K1042, I267-K1042, D268-K1042, V269-K1042, I270-K1042, S271-K1042,E272-K1042, K273-K1042, T274-K1042, L275-K1042, R276-K1042, N277-K1042,T278-K1042, V279-K1042, T280-K1042, L281-K1042, T282-K1042, A283-K1042,G284-K1042, R285-K1042, G286-K1042, R287-K1042, G288-K1042, K289-K1042,S290-K1042, A291-K1042, A292-K1042, L293-K1042, G294-K1042, I295-K1042,A296-K1042, I297-K1042, A298-K1042, A299-K1042, A300-K1042, I301-K1042,S302-K1042, H303-K1042, G304-K1042, Y305-K1042, S306-K1042, N307-K1042,I308-K1042, F309-K1042, V310-K1042, T311-K1042, S312-K1042, P313-K1042,S314-K1042, P315-K1042, E316-K1042, N317-K1042, L318-K1042, K319-K1042,T320-K1042, L321-K1042, F322-K1042, E323-K1042, F324-K1042, I325-K1042,F326-K1042, K327-K1042, G328-K1042, F329-K1042, D330-K1042, A331-K1042,L332-K1042, G333-K1042, Y334-K1042, T335-K1042, E336-K1042, H337-K1042,M338-K1042, D339-K1042, Y340-K1042, D341-K1042, I342-K1042, I343-K1042,Q344-K1042, S345-K1042, T346-K1042, N347-K1042, P348-K1042, S349-K1042,F350-K1042, N351-K1042, K352-K1042, A353-K1042, I354-K1042, V355-K1042,R356-K1042, V357-K1042, D358-K1042, V359-K1042, K360-K1042, R361-K1042,E362-K1042, H363-K1042, R364-K1042, Q365-K1042, T366-K1042, I367-K1042,Q368-K1042, Y369-K1042, I370-K1042, S371-K1042, P372-K1042, N373-K1042,D374-K1042, S375-K1042, H376-K1042, V377-K1042, L378-K1042, G379-K1042,Q380-K1042, A381-K1042, E382-K1042, L383-K1042, L384-K1042, I385-K1042,I386-K1042, D387-K1042, E388-K1042, A389-K1042, A390-K1042, A391-K1042,I392-K1042, P393-K1042, L394-K1042, P395-K1042, I396-K1042, V397-K1042,K398-K1042, K399-K1042, L400-K1042, M401-K1042, G402-K1042, P403-K1042,Y404-K1042, L405-K1042, I406-K1042, F407-K1042, M408-K1042, A409-K1042,S410-K1042, T411-K1042, I412-K1042, N413-K1042, G414-K1042, Y415-K1042,E416-K1042, G417-K1042, T418-K1042, G419-K1042, R420-K1042, S421-K1042,L422-K1042, S423-K1042, L424-K1042, K425-K1042, L426-K1042, I427-K1042,Q428-K1042, Q429-K1042, L430-K1042, R431-K1042, T432-K1042, Q433-K1042,S434-K1042, N435-K1042, N436-K1042, A437-K1042, T438-K1042, P439-K1042,S440-K1042, E441-K1042, T442-K1042, T443-K1042, V444-K1042, V445-K1042,S446-K1042, R447-K1042, D448-K1042, K449-K1042, K450-K1042, S451-K1042,N452-K1042, E453-K1042, I454-K1042, T455-K1042, G456-K1042, A457-K1042,L458-K1042, T459-K1042, R460-K1042, T461-K1042, L462-K1042, K463-K1042,E464-K1042, V465-K1042, V466-K1042, L467-K1042, D468-K1042, E469-K1042,P470-K1042, I471-K1042, R472-K1042, Y473-K1042, A474-K1042, P475-K1042,G476-K1042, D477-K1042, P478-K1042, I479-K1042, E480-K1042, K481-K1042,W482-K1042, L483-K1042, N484-K1042, K485-K1042, L486-K1042, L487-K1042,C488-K1042, L489-K1042, D490-K1042, V491-K1042, S492-K1042, L493-K1042,S494-K1042, K495-K1042, N496-K1042, A497-K1042, K498-K1042, F499-K1042,A500-K1042, T501-K1042, K502-K1042, G503-K1042, T504-K1042, P505-K1042,H506-K1042, P507-K1042, S508-K1042, Q509-K1042, C510-K1042, Q511-K1042,L512-K1042, F513-K1042, Y514-K1042, V515-K1042, N516-K1042, R517-K1042,D518-K1042, T519-K1042, L520-K1042, F521-K1042, S522-K1042, Y523-K1042,H524-K1042, P525-K1042, V526-K1042, S527-K1042, E528-K1042, A529-K1042,F530-K1042, L531-K1042, Q532-K1042, K533-K1042, M534-K1042, M535-K1042,A536-K1042, L537-K1042, Y538-K1042, V539-K1042, A540-K1042, S541-K1042,H542-K1042, Y543-K1042, K544-K1042, N545-K1042, S546-K1042, P547-K1042,N548-K1042, D549-K1042, L550-K1042, Q551-K1042, L552-K1042, M553-K1042,S554-K1042, D555-K1042, A556-K1042, P557-K1042, A558-K1042, H559-K1042,Q560-K1042, L561-K1042, F562-K1042, V563-K1042, L564-K1042, L565-K1042,P566-K1042, P567-K1042, I568-K1042, E569-K1042, A570-K1042, G571-K1042,D572-K1042, N573-K1042, R574-K1042, V575-K1042, P576-K1042, D577-K1042,P578-K1042, L579-K1042, C580-K1042, V581-K1042, I582-K1042, Q583-K1042,L584-K1042, A585-K1042, L586-K1042, E587-K1042, G588-K1042, E589-K1042,I590-K1042, S591-K1042, K592-K1042, E593-K1042, S594-K1042, V595-K1042,R596-K1042, K597-K1042, S598-K1042, L599-K1042, S600-K1042, R601-K1042,G602-K1042, Q603-K1042, R604-K1042, A605-K1042, G606-K1042, G607-K1042,D608-K1042, L609-K1042, I610-K1042, P611-K1042, W612-K1042, L613-K1042,I614-K1042, S615-K1042, Q616-K1042, Q617-K1042, F618-K1042, Q619-K1042,D620-K1042, E621-K1042, E622-K1042, F623-K1042, A624-K1042, S625-K1042,L626-K1042, S627-K1042, G628-K1042, A629-K1042, R630-K1042, V631-K1042,V632-K1042, R633-K1042, I634-K1042, A635-K1042, T636-K1042, N637-K1042,P638-K1042, E639-K1042, Y640-K1042, S641-K1042, G642-K1042, M643-K1042,G644-K1042, Y645-K1042, G646-K1042, S647-K1042, R648-K1042, A649-K1042,M650-K1042, E651-K1042, L652-K1042, L653-K1042, R654-K1042, D655-K1042,Y656-K1042, Y657-K1042, S658-K1042, G659-K1042, K660-K1042, F661-K1042,T662-K1042, D663-K1042, I664-K1042, S665-K1042, E666-K1042, S667-K1042,T668-K1042, E669-K1042, L670-K1042, N671-K1042, D672-K1042, H673-K1042,T674-K1042, I675-K1042, T676-K1042, R677-K1042, V678-K1042, T679-K1042,D680-K1042, S681-K1042, E682-K1042, L683-K1042, A684-K1042, N685-K1042,A686-K1042, S687-K1042, L688-K1042, K689-K1042, D690-K1042, E691-K1042,I692-K1042, K693-K1042, L694-K1042, R695-K1042, D696-K1042, V697-K1042,K698-K1042, T699-K1042, L700-K1042, P701-K1042, P702-K1042, L703-K1042,L704-K1042, L705-K1042, K706-K1042, L707-K1042, S708-K1042, E709-K1042,K710-K1042, A711-K1042, P712-K1042, Y713-K1042, Y714-K1042, L715-K1042,H716-K1042, Y717-K1042, L718-K1042, G719-K1042, V720-K1042, S721-K1042,Y722-K1042, G723-K1042, F724-K1042, T725-K1042, S726-K1042, Q727-K1042,L728-K1042, H729-K1042, K730-K1042, F731-K1042, W732-K1042, K733-K1042,K734-K1042, A735-K1042, G736-K1042, F737-K1042, T738-K1042, P739-K1042,V740-K1042, Y741-K1042, L742-K1042, R743-K1042, Q744-K1042, T745-K1042,P746-K1042, N747-K1042, E748-K1042, L749-K1042, T750-K1042, G751-K1042,E752-K1042, H753-K1042, T754-K1042, S755-K1042, V756-K1042, V757-K1042,I758-K1042, S759-K1042, V760-K1042, L761-K1042, P762-K1042, G763-K1042,R764-K1042, E765-K1042, D766-K1042, K767-K1042, W768-K1042, L769-K1042,H770-K1042, E771-K1042, F772-K1042, S773-K1042, K774-K1042, D775-K1042,F776-K1042, H777-K1042, K778-K1042, R779-K1042, F780-K1042, L781-K1042,S782-K1042, L783-K1042, L784-K1042, S785-K1042, Y786-K1042, E787-K1042,F788-K1042, K789-K1042, K790-K1042, F791-K1042, Q792-K1042, A793-K1042,S794-K1042, Q795-K1042, A796-K1042, L797-K1042, S798-K1042, I799-K1042,I800-K1042, E801-K1042, A802-K1042, A803-K1042, E804-K1042, Q805-K1042,G806-K1042, E807-K1042, G808-K1042, D809-K1042, E810-K1042, T811-K1042,T812-K1042, S813-K1042, Q814-K1042, K815-K1042, L816-K1042, T817-K1042,K818-K1042, E819-K1042, Q820-K1042, L821-K1042, D822-K1042, L823-K1042,L824-K1042, L825-K1042, S826-K1042, P827-K1042, F828-K1042, D829-K1042,L830-K1042, K831-K1042, R832-K1042, L833-K1042, D834-K1042, S835-K1042,Y836-K1042, A837-K1042, N838-K1042, N839-K1042, L840-K1042, L841-K1042,D842-K1042, Y843-K1042, H844-K1042, V845-K1042, I846-K1042, V847-K1042,D848-K1042, M849-K1042, L850-K1042, P851-K1042, L852-K1042, I853-K1042,S854-K1042, Q855-K1042, L856-K1042, F857-K1042, F858-K1042, S859-K1042,K860-K1042, K861-K1042, T862-K1042, G863-K1042, Q864-K1042, D865-K1042,I866-K1042, S867-K1042, L868-K1042, S869-K1042, S870-K1042, V871-K1042,Q872-K1042, S873-K1042, A874-K1042, I875-K1042, L876-K1042, L877-K1042,A878-K1042, I879-K1042, G880-K1042, L881-K1042, Q882-K1042, H883-K1042,K884-K1042, D885-K1042, M886-K1042, D887-K1042, Q888-K1042, I889-K1042,A890-K1042, K891-K1042, E892-K1042, L893-K1042, N894-K1042, L895-K1042,P896-K1042, T897-K1042, N898-K1042, Q899-K1042, A900-K1042, M901-K1042,A902-K1042, M903-K1042, F904-K1042, A905-K1042, K906-K1042, I907-K1042,I908-K1042, R909-K1042, K910-K1042, F911-K1042, S912-K1042, T913-K1042,Y914-K1042, F915-K1042, R916-K1042, K917-K1042, V918-K1042, L919-K1042,S920-K1042, K921-K1042, A922-K1042, I923-K1042, E924-K1042, E925-K1042,S926-K1042, M927-K1042, P928-K1042, D929-K1042, L930-K1042, E931-K1042,D932-K1042, E933-K1042, N934-K1042, V935-K1042, D936-K1042, A937-K1042,M938-K1042, N939-K1042, G940-K1042, K941-K1042, E942-K1042, T943-K1042,E944-K1042, Q945-K1042, I946-K1042, D947-K1042, Y948-K1042, K949-K1042,A950-K1042, I951-K1042, E952-K1042, Q953-K1042, K954-K1042, L955-K1042,Q956-K1042, D957-K1042, D958-K1042, L959-K1042, E960-K1042, E961-K1042,A962-K1042, G963-K1042, D964-K1042, E965-K1042, A966-K1042, I967-K1042,K968-K1042, E969-K1042, M970-K1042, R971-K1042, E972-K1042, K973-K1042,Q974-K1042, R975-K1042, E976-K1042, L977-K1042, I978-K1042, N979-K1042,A980-K1042, L981-K1042, N982-K1042, L983-K1042, D984-K1042, K985-K1042,Y986-K1042, A987-K1042, I988-K1042, A989-K1042, E990-K1042, D991-K1042,A992-K3 1042, E993-K1042, W994-K1042, D995-K1042, E996-K1042,K997-K1042, S998-K1042, M999-K1042, D1000-K1042, K1001-K1042,A1002-K1042, T1003-K1042, K1004-K1042, G1005-K1042, K1006-K1042,G1007-K1042, N1008-K1042, V1009-K1042, V1010-K1042, S1011-K1042,I1012-K1042, K1013-K1042, S1014-K1042, G1015-K1042, K1016-K1042,R1017-K1042, K1018-K1042, S1019-K1042, K1020-K1042, E1021-K1042,N1022-K1042, A1023-K1042, N1024-K1042, D1025-K1042, I1026-K1042,Y1027-K1042, E1028-K1042, K1029-K1042, E1030-K1042, M1031-K1042,K1032-K1042, A1033-K1042, V1034-K1042, K1035-K1042, and/or K1036-K1042of SEQ ID NO:16. Polynucleotide sequences encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese N-terminal CaYNL132w deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYNL132w deletionpolypeptides are encompassed by the present invention: M1-K1042,M1-K1041, M1-S1040, M1-K1039, M1-K1038, M1-S1037, M1-K1036, M1-K1035,M1-V1034, M1-A1033, M1-K1032, M1-M1031, M1-E1030, M1-K1029, M1-E1028,M1-Y1027, M1-I1026, M1-D1025, M1-N1024, M1-A1023, M1-N1022, M1-E1021,M1-K1020, M1-S1019, M1-K1018, M1-R1017, M1-K1016, M1-G1015, M1-S1014,M1-K1013, M1-I1012, M1-S1011, M1-V1010, M1-V1009, M1-N1008, M1-G1007,M1-K1006, M1-G1005, M1-K1004, M1-T1003, M1-A1002, M1-K1001, M1-D1000,M1-M999, M1-S998, M1-K997, M1-E996, M1-D995, M1-W994, M1-E993, M1-A992,M1-D991, M1-E990, M1-A989, M1-I988, M1-A987, M1-Y986, M1-K985, M1-D984,M1-L983, M1-N982, M1-L981, M1-A980, M1-N979, M1-I978, M1-L977, M1-E976,M1-R975, M1-Q974, M1-K973, M1-E972, M1-R971, M1-M970, M1-E969, M1-K968,M1-I967, M1-A966, M1-E965, M1-D964, M1-G963, M1-A962, M1-E961, M1-E960,M1-L959, M1-D958, M1-D957, M1-Q956, M1-L955, M1-K954, M1-Q953, M1-E952,M1-I951, M1-A950, M1-K949, M1-Y948, M1-D947, M1-I946, M1-Q945, M1-E944,M1-T943, M1-E942, M1-K941, M1-G940, M1-N939, M1-M938, M1-A937, M1-D936,M1-V935, M1-N934, M1-E933, M1-D932, M1-E931, M1-L930, M1-D929, M1-P928,M1-M927, M1-S926, M1-E925, M1-E924, M1-I923, M1-A922, M1-K921, M1-S920,M1-L919, M1-V918, M1-K917, M1-R916, M1-F915, M1-Y914, M1-T913, M1-S912,M1-F911, M1-K910, M1-R909, M1-I908, M1-I907, M1-K906, M1-A905, M1-F904,M1-M903, M1-A902, M1-M901, M1-A900, M1-Q899, M1-N898, M1-T897, M1-P896,M1-L895, M1-N894, M1-L893, M1-E892, M1-K891, M1-A890, M1-I889, M1-Q888,M1-D887, M1-M886, M1-D885, M1-K884, M1-H883, M1-Q882, M1-L881, M1-G880,M1-I879, M1-A878, M1-L877, M1-L876, M1-I875, M1-A874, M1-S873, M1-Q872,M1-V871, M1-S870, M1-S869, M1-L868, M1-S867, M1-I866, M1-D865, M1-Q864,M1-G863, M1-T862, M1-K861, M1-K860, M1-S859, M1-F858, M1-F857, M1-L856,M1-Q855, M1-S854, M1-I853, M1-L852, M1-P851, M1-L850, M1-M849, M1-D848,M1-V847, M1-I846, M1-V845, M1-H844, M1-Y843, M1-D842, M1-L841, M1-L840,M1-N839, M1-N838, M1-A837, M1-Y836, M1-S835, M1-D834, M1-L833, M1-R832,M1-K831, M1-L830, M1-D829, M1-F828, M1-P827, M1-S826, M1-L825, M1-L824,M1-L823, M1-D822, M1-L821, M1-Q820, M1-E819, M1-K818, M1-T817, M1-L816,M1-K815, M1-Q814, M1-S813, M1-T812, M1-T811, M1-E810, M1-D809, M1-G808,M1-E807, M1-G806, M1-Q805, M1-E804, M1-A803, M1-A802, M1-E801, M1-I800,M1-I799, M1-S798, M1-L797, M1-A796, M1-Q795, M1-S794, M1-A793, M1-Q792,M1-F791, M1-K790, M1-K789, M1-F788, M1-E787, M1-Y786, M1-S785, M1-L784,M1-L783, M1-S782, M1-L781, M1-F780, M1-R779, M1-K778, M1-H777, M1-F776,M1-D775, M1-K774, M1-S773, M1-F772, M1-E771, M1-H770, M1-L769, M1-W768,M1-K767, M1-D766, M1-E765, M1-R764, M1-G763, M1-P762, M1-L761, M1-V760,M1-S759, M1-I758, M1-V757, M1-V756, M1-S755, M1-T754, M1-H753, M1-E752,M1-G751, M1-T750, M1-L749, M1-E748, M1-N747, M1-P746, M1-T745, M1-Q744,M1-R743, M1-L742, M1-Y741, M1-V740, M1-P739, M1-T738, M1-F737, M1-G736,M1-A735, M1-K734, M1-K733, M1-W732, M1-F731, M1-K730, M1-H729, M1-L728,M1-Q727, M1-S726, M1-T725, M1-F724, M1-G723, M1-Y722, M1-S721, M1-V720,M1-G719, M1-L718, M1-Y717, M1-H716, M1-L715, M1-Y714, M1-Y713, M1-P712,M1-A711, M1-K710, M1-E709, M1-S708, M1-L707, M1-K706, M1-L705, M1-L704,M1-L703, M1-P702, M1-P701, M1-L700, M1-T699, M1-K698, M1-V697, M1-D696,M1-R695, M1-L694, M1-K693, M1-I692, M1-E691, M1-D690, M1-K689, M1-L688,M1-S687, M1-A686, M1-N685, M1-A684, M1-L683, M1-E682, M1-S681, M1-D680,M1-T679, M1-V678, M1-R677, M1-T676, M1-I675, M1-T674, M1-H673, M1-D672,M1-N671, M1-L670, M1-E669, M1-T668, M1-S667, M1-E666, M1-S665, M1-I664,M1-D663, M1-T662, M1-F661, M1-K660, M1-G659, M1-S658, M1-Y657, M1-Y656,M1-D655, M1-R654, M1-L653, M1-L652, M1-E651, M1-M650, M1-A649, M1-R648,M1-S647, 1-G646, M1-Y645, M1-G644, M1-M643, M1-G642, M1-S641, M1-Y640,M1-E639, M1-P638, M1-N637, M1-T636, M1-A635, M1-I634, M1-R633, M1-V632,M1-V631, M1-R630, M1-A629, M1-G628, M1-S627, M1-L626, M1-S625, M1-A624,M1-F623, M1-E622, M1-E621, M1-D620, M1-Q619, M1-F618, M1-Q617, M1-Q616,M1-S615, M11-614, M1-L613, M1-W612, M1-P611, M1-I610, M1-L609, M1-D608,M1-G607, M1-G606, M1-A605, M1-R604, M1-Q603, M1-G602, M1-R601, M1-S600,M1-L599, M1-S598, M1-K597, M1-R596, M1-V595, M1-S594, M1-E593, M1-K592,M1-S591, M1-I590, M1-E589, M1-G588, M1-E587, M1-L586, M1-A585, M1-L584,M1-Q583, M1-I582, M1-V581, M1-C580, M1-L579, M1-P578, M1-D577, M1-P576,M1-V575, M1-R574, M1-N573, M1-D572, M1-G571, M1-A570, M1-E569, M1-I568,M1-P567, M1-P566, M1-L565, M1-L564, M1-V563, M1-F562, M1-L561, M1-Q560,M1-H559, M1-A558, M1-P557, M1-A556, M1-D555, M1-S554, M1-M553, M1-L552,M1-Q551, M1-L550, M1-D549, M1-N548, M1-P547, M1-S546, M1-N545, M1-K544,M1-Y543, M1-H542, M1-S541, M1-A540, M1-V539, M1-Y538, M1-L537, M1-A536,M1-M535, M1-M534, M1-K533, M1-Q532, M1-L531, M1-F530, M1-A529, M1-E528,M1-S527, M1-V526, M1-P525, M1-H524, M1-Y523, M1-S522, M1-F521, M1-L520,M1-T519, M1-D518, M1-R517, M1-N516, M1-V515, M1-Y514, M1-F513, M1-L512,M1-Q511, M1-C510, M1-Q509, M1-S508, M1-P507, M1-H506, M1-P505, M1-T504,M1-G503, M1-K502, M1-T501, M1-A500, M1-F499, M1-K498, M1-A497, M1-N496,M1-K495, M1-S494, M1-L493, M1-S492, M1-V491, M1-D490, M1-L489, M1-C488,M1-L487, M1-L486, M1-K485, M1-N484, M1-L483, M1-W482, M1-K481, M1-E480,M1-I479, M1-P478, M1-D477, M1-G476, M1-P475, M1-A474, M1-Y473, M1-R472,M1-I471, M1-P470, M1-E469, M1-D468, M1-L467, M1-V466, M1-V465, M1-E464,M1-K463, M1-L462, M1-T461, M1-R460, M1-T459, M1-L458, M1-A457, M1-G456,M1-T455, M1-I454, M1-E453, M1-N452, M1-S451, M1-K450, M1-K449, M1-D448,M1-R447, M1-S446, M1-V445, M1-V444, M1-T443, M1-T442, M1-E441, M1-S440,M1-P439, M1-T438, M1-A437, M1-N436, M1-N435, M1-S434, M1-Q433, M1-T432,M1-R431, M1-L430, M1-Q429, M1-Q428, M1-I427, M1-L426, M1-K425, M1-L424,M1-S423, M1-L422, M1-S421, M1-R420, M1-G419, M1-T418, M1-G417, M1-E416,M1-Y415, M1-G414, M1-N413, M1-I412, M1-T411, M1-S410, M1-A409, M1-M408,M1-F407, M1-I406, M1-L405, M1-Y404, M1-P403, M1-G402, M1-M401, M1-L400,M1-K399, M1-K398, M1-V397, M1-I396, M1-P395, M1-L394, M1-P393, M1-I392,M1-A391, M1-A390, M1-A389, M1-E388, M1-D387, M1-I386, M1-I385, M1-L384,M1-L383, M1-E382, M1-A381, M1-Q380, M1-G379, M1-L378, M1-V377, M1-H376,M1-S375, M1-D374, M1-N373, M1-P372, M1-S371, M1-I370, M1-Y369, M1-Q368,M1-I367, M1-T366, M1-Q365, M1-R364, M1-H363, M1-E362, M1-R361, M1-K360,M1-V359, M1-D358, M1-V357, M1-R356, M1-V355, M1-I354, M1-A353, M1-K352,M1-N351, M1-F350, M1-S349, M1-P348, M1-N347, M1-T346, M1-S345, M1-Q344,M1-I343, M1-I342, M1-D341, M1-Y340, M1-D339, M1-M338, M1-H337, M1-E336,M1-T335, M1-Y334, M1-G333, M1-L332, M1-A331, M1-D330, M1-F329, M1-G328,M1-K327, M1-F326, M1-I325, M1-F324, M1-E323, M1-F322, M1-L321, M1-T320,M1-K319, M1-L318, M1-N317, M1-E316, M1-P315, M1-S314, M1-P313, M1-S312,M1-T311, M1-V310, M1-F309, M1-I308, M1-N307, M1-S306, M1-Y305, M1-G304,M1-H303, M1-S302, M1-I301, M1-A300, M1-A299, M1-A298, M1-I297, M1-A296,M1-I295, M1-G294, M1-L293, M1-A292, M1-A291, M1-S290, M1-K289, M1-G288,M1-R287, M1-G286, M1-R285, M1-G284, M1-A283, M1-T282, M1-L281, M1-T280,M1-V279, M1-T278, M1-N277, M1-R276, M1-L275, M1-T274, M1-K273, M1-E272,M1-S271, M1-I270, M1-V269, M1-D268, M1-I267, M1-F266, M1-T265, M1-L264,M1-I263, M1-A262, M1-Q261, M1-A260, M1-Q259, M1-N258, M1-I257, M1-T256,M1-K255, M1-S254, M1-L253, M1-A252, M1-V251, M1-L250, M1-S249, M1-G248,M1-A247, M1-P246, M1-Q245, M1-V244, M1-D243, M1-A242, M1-L241, M1-S240,M1-E239, M1-K238, M1-L237, M1-E236, M1-K235, M1-L234, M1-E233, M1-K232,M1-A231, M1-N230, M1-P229, M1-T228, M1-L227, M1-E226, M1-D225, M1-D224,M1-D223, M1-K222, M1-P221, M1-P220, M1-L219, M1-P218, M1-K217, M1-V216,M1-H215, M1-K214, M1-G213, M1-G212, M1-S211, M1-I210, M1-P209, M1-L208,M1-V207, M1-N206, M1-L205, M1-E204, M1-D203, M1-D202, M1-V201, M1-V200,M1-L199, M1-C198, M1-N197, M1-E196, M1-C195, M1-S194, M1-G193, M1-L192,M1-S191, M1-L190, M1-L189, M1-F188, M1-R187, M1-E186, M1-N185, M1-F184,M1-R183, M1-A182, M1-V181, M1-V180, M1-D179, M1-D178, M1-H177, M1-A176,M1-E175, M1-T174, M1-R173, M1-Y172, M1-R171, M1-S170, M1-H169, M1-I168,M1-D167, M1-M166, M1-S165, M1-M164, M1-T163, M1-Y162, M1-L161, M1-Q160,M1-K159, M1-L158, M1-S157, M1-T156, M1-M155, M1-N154, M1-K153, M1-L152,M1-L151, M1-I150, M1-V149, M1-V148, M1-L147, M1-G146, M1-G145, M1-G144,M1-E143, M1-V142, M1-T141, M1-E140, M1-I139, M1-T138, M1-R137, M1-A136,M1-L135, M1-L134, M1-N133, M1-P132, M1-T131, M1-I130, M1-A129, M1-E128,M1-F127, M1-D126, M1-Q125, M1-L124, M1-I123, M1-C122, M1-M121, M1-G120,M1-Y119, M1-T118, M1-N117, M1-G116, M1-L115, M1-I114, M1-K113, M1-E112,M1-T111, M1-E110, M1-K109, M1-Y108, M1-Y107, M1-V106, M1-Y105, M1-R104,M1-I103, M1-H102, M1-Q101, M1-N100, M1-S99, M1-I98, M1-F97, M1-A96,M1-E95, M1-F94, M1-P93, M1-D92, M1-Q91, M1-E90, M1-N89, M1-V88, M1-E87,M1-R86, M1-I85, M1-G84, M1-R83, M1-K82, M1-I81, M1-D80, M1-K79, M1-K78,M1-I77, M1-K76, M1-A75, M1-E74, M1-R73, M1-K72, M1-Q71, M1-R70, M1-H69,M1-S68, M1-T67, M1-F66, M1-G65, M1-L64, M1-L63, M1-K62, M1-K61, M1-K60,M1-Y59, M1-A58, M1-W57, M1-L56, M1-V55, M1-S54, M1-K53, M1-N52, M1-M51,M1-K50, M1-L49, M1-D48, M1-A47, M1-S46, M1-M45, M1-M44, M1-L43, M1-Y42,M1-H41, M1-L40, M1-N39, M1-P38, M1-L37, M1-Q36, M1-N35, M1-R34, M1-A33,M1-K32, M1-D31, M1-G30, M1-V29, M1-I28, M1-I27, M1-F26, M1-F25, M1-S24,M1-R23, M1-Q22, M1-K21, M1-E20, M1-Q19, M1-V18, M1-G17, M1-N16, M1-R15,M1-I14, M1-L13, M1-A12, M1-P11, M1-I10, M1-R9, M1-A8, and/or M1-D7 ofSEQ ID NO:16. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseC-terminal CaYNL132w deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:5 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 3112 ofSEQ ID NO:5, b is an integer between 15 to 3126, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:5,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:6

The polynucleotide sequence (SEQ ID NO:6) and deduced amino acidsequence (SEQ ID NO:17) of the novel fungal essential gene, CaYGR145w(also referred to as FCG12), of the present invention. The CaYGR145wpolypeptide (SEQ ID NO:17) is encoded by nucleotides 1 to 2250 of SEQ IDNO:6 and has a predicted molecular weight of 85.0 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYGR145w. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 2250 of SEQ ID NO:6, and the polypeptide corresponding to aminoacids 2 thru 1042 of SEQ ID NO:18. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYGR145w polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYGR145w deletionpolypeptides are encompassed by the present invention: M1-M750, V2-M750,L3-M750, K4-M750, S5-M750, T6-M750, T7-M750, A8-M750, G9-M750, N10-M750,V11-M750, S12-M750, V13-M750, Y14-M750, Q15-M750, V16-M750, S17-M750,G18-M750, T19-M750, N20-M750, V21-M750, S22-M750, R23-M750, S24-M750,L25-M750, P26-M750, D27-M750, W28-M750, I29-M750, D30-M750, K31-M750,K32-M750, R33-M750, K34-M750, R35-M750, A36-M750, L37-M750, K38-M750,H39-M750, D40-M750, L41-M750, E42-M750, Y43-M750, Q44-M750, N45-M750,R46-M750, I47-M750, E48-M750, L49-M750, I50-M750, Q51-M750, D52-M750,F53-M750, E54-M750, F55-M750, S56-M750, E57-M750, A58-M750, S59-M750,N60-M750, K61-M750, I62-M750, K63-M750, V64-M750, T65-M750, N66-M750,D67-M750, G68-M750, Q69-M750, Y70-M750, C71-M750, M72-M750, A73-M750,T74-M750, G75-M750, T76-M750, Y77-M750, K78-M750, P79-M750, Q80-M750,I81-M750, H82-M750, V83-M750, Y84-M750, E85-M750, F86-M750, A87-M750,N88-M750, L89-M750, S90-M750, L91-M750, K92-M750, F93-M750, D94-M750,R95-M750, H96-M750, T97-M750, N98-M750, V99-M750, E100-M750, N101-M750,I102-M750, D103-M750, F104-M750, L105-M750, I106-M750, L107-M750,S108-M750, N109-M750, D110-M750, W111-M750, T112-M750, K113-M750,S114-M750, V115-M750, H116-M750, L117-M750, Q118-M750, C119-M750,D120-M750, R121-M750, S122-M750, I123-M750, E124-M750, F125-M750,Q126-M750, T127-M750, A128-M750, G129-M750, G130-M750, V131-M750,H132-M750, Y133-M750, R134-M750, T135-M750, R136-M750, I137-M750,P138-M750, K139-M750, F140-M750, G141-M750, R142-M750, C143-M750,L144-M750, T145-M750, Y146-M750, N147-M750, P148-M750, I149-M750,N150-M750, C151-M750, D152-M750, L153-M750, I154-M750, V155-M750,G156-M750, S157-M750, S158-M750, S159-M750, D160-M750, E161-M750,L162-M750, Y163-M750, R164-M750, L165-M750, N166-M750, L167-M750,D168-M750, Q169-M750, G170-M750, R171-M750, F172-M750, L173-M750,S174-M750, P175-M750, L176-M750, K177-M750, L178-M750, D179-M750,M180-M750, T181-M750, D182-M750, G183-M750, G184-M750, N185-M750,I186-M750, D187-M750, S188-M750, G189-M750, C190-M750, N191-M750,A192-M750, V193-M750, D194-M750, I195-M750, N196-M750, S197-M750,M198-M750, H199-M750, G200-M750, L201-M750, I202-M750, S203-M750,A204-M750, G205-M750, L206-M750, D207-M750, D208-M750, G209-M750,T210-M750, V211-M750, E212-M750, F213-M750, W214-M750, D215-M750,P216-M750, R217-M750, S218-M750, K219-M750, Q220-M750, R221-M750,A222-M750, G223-M750, K224-M750, L225-M750, F226-M750, V227-M750,S228-M750, D229-M750, Q230-M750, L231-M750, I232-M750, N233-M750,S234-M750, T235-M750, N236-M750, N237-M750, T238-M750, E239-M750,Q240-M750, S241-M750, S242-M750, C243-M750, G244-M750, I245-M750,T246-M750, S247-M750, L248-M750, A249-M750, F250-M750, R251-M750,P252-M750, Q253-M750, D254-M750, A255-M750, L256-M750, N257-M750,F258-M750, A259-M750, C260-M750, G261-M750, T262-M750, S263-M750,N264-M750, G265-M750, Q266-M750, T267-M750, L268-M750, L269-M750,Y270-M750, D271-M750, L272-M750, R273-M750, A274-M750, S275-M750,E276-M750, P277-M750, Y278-M750, Q279-M750, I280-M750, K281-M750,D282-M750, Q283-M750, G284-M750, Y285-M750, G286-M750, Y287-M750,D288-M750, I289-M750, K290-M750, K291-M750, I292-M750, I293-M750,W294-M750, C295-M750, Q296-M750, D297-M750, S298-M750, L299-M750,K300-M750, P301-M750, E302-M750, M303-M750, I304-M750, L305-M750,T306-M750, S307-M750, D308-M750, K309-M750, R310-M750, I311-M750,V312-M750, K313-M750, I314-M750, W315-M750, D316-M750, H317-M750,T318-M750, N319-M750, G320-M750, K321-M750, S322-M750, F323-M750,A324-M750, S325-M750, M326-M750, E327-M750, P328-M750, T329-M750,V330-M750, D331-M750, I332-M750, N333-M750, D334-M750, I335-M750,C336-M750, H337-M750, I338-M750, P339-M750, Q340-M750, S341-M750,G342-M750, M343-M750, F344-M750, F345-M750, M346-M750, A347-M750,N348-M750, E349-M750, G350-M750, M351-M750, P352-M750, M353-M750,H354-M750, T355-M750, Y356-M750, Y357-M750, I358-M750, P359-M750,N360-M750, L361-M750, G362-M750, S363-M750, A364-M750, P365-M750,N366-M750, W367-M750, C368-M750, S369-M750, F370-M750, L371-M750,D372-M750, N373-M750, V374-M750, T375-M750, E376-M750, E377-M750,L378-M750, E379-M750, E380-M750, K381-M750, P382-M750, S383-M750,N384-M750, S385-M750, I386-M750, Y387-M750, P388-M750, T389-M750,F390-M750, K391-M750, F392-M750, I393-M750, T394-M750, R395-M750,D396-M750, E397-M750, M398-M750, V399-M750, K400-M750, L401-M750,N402-M750, L403-M750, T404-M750, H405-M750, L406-M750, I407-M750,G408-M750, I409-M750, K410-M750, V411-M750, L412-M750, R413-M750,S414-M750, Y415-M750, M416-M750, H417-M750, G418-M750, F419-M750,F420-M750, I421-M750, N422-M750, T423-M750, E424-M750, L425-M750,Y426-M750, D427-M750, K428-M750, V429-M750, N430-M750, L431-M750,I432-M750, S433-M750, N434-M750, P435-M750, N436-M750, S437-M750,I438-M750, Y439-M750, D440-M750, Q441-M750, R442-M750, K443-M750,R444-M750, E445-M750, I446-M750, A447-M750, N448-M750, K449-M750,I450-M750, N451-M750, E452-M750, E453-M750, R454-M750, K455-M750,S456-M750, R457-M750, I458-M750, L459-M750, T460-M750, S461-M750,S462-M750, N463-M750, G464-M750, N465-M750, D466-M750, L467-M750,P468-M750, T469-M750, K470-M750, I471-M750, K472-M750, V473-M750,N474-M750, K475-M750, D476-M750, L477-M750, V478-M750, N479-M750,K480-M750, L481-M750, Q482-M750, T483-M750, K484-M750, F485-M750,A486-M750, E487-M750, N488-M750, G489-M750, T490-M750, P491-M750,D492-M750, G493-M750, N494-M750, A495-M750, N496-M750, G497-M750,A498-M750, T499-M750, D500-M750, Y501-M750, V502-M750, E503-M750,S504-M750, I505-M750, V506-M750, N507-M750, D508-M750, D509-M750,R510-M750, F511-M750, R512-M750, E513-M750, M514-M750, F515-M750,E516-M750, N517-M750, P518-M750, D519-M750, F520-M750, E521-M750,I522-M750, D523-M750, E524-M750, E525-M750, S526-M750, H527-M750,E528-M750, Y529-M750, K530-M750, Q531-M750, L532-M750, N533-M750,P534-M750, V535-M750, K536-M750, S537-M750, T538-M750, K539-M750,D540-M750, I541-M750, T542-M750, T543-M750, T544-M750, N545-M750,T546-M750, G547-M750, T548-M750, T549-M750, N550-M750, S551-M750,R552-M750, G553-M750, R554-M750, G555-M750, L556-M750, T557-M750,A558-M750, A559-M750, E560-M750, E561-M750, S562-M750, D563-M750,E564-M750, E565-M750, R566-M750, L567-M750, N568-M750, M569-M750,K570-M750, D571-M750, S572-M750, H573-M750, H574-M750, T575-M750,G576-M750, L577-M750, D578-M750, S579-M750, D580-M750, E581-M750,S582-M750, D583-M750, E584-M750, E585-M750, S586-M750, D587-M750,S588-M750, E589-M750, S590-M750, E591-M750, E592-M750, Q593-M750,S594-M750, E595-M750, D596-M750, E597-M750, A598-M750, K599-M750,S600-M750, A601-M750, E602-M750, T603-M750, R604-M750, E605-M750,R606-M750, V607-M750, G608-M750, K609-M750, E610-M750, L611-M750,N612-M750, K613-M750, I614-M750, R615-M750, Q616-M750, S617-M750,K618-M750, Q619-M750, K620-M750, Q621-M750, Q622-M750, Q623-M750,Q624-M750, D625-M750, S626-M750, K627-M750, K628-M750, F629-M750,Q630-M750, N631-M750, E632-M750, M633-M750, K634-M750, I635-M750,L636-M750, S637-M750, Q638-M750, Q639-M750, S640-M750, S641-M750,S642-M750, S643-M750, S644-M750, S645-M750, S646-M750, L647-M750,A648-M750, N649-M750, T650-M750, E651-M750, K652-M750, A653-M750,S654-M750, V655-M750, S656-M750, F657-M750, G658-M750, S659-M750,Q660-M750, V661-M750, N662-M750, K663-M750, L664-M750, N665-M750,K666-M750, I667-M750, S668-M750, K669-M750, Q670-M750, N671-M750,K672-M750, N673-M750, N674-M750, N675-M750, S676-M750, I677-M750,S678-M750, N679-M750, A680-M750, K681-M750, D682-M750, A683-M750,R684-M750, L685-M750, R686-M750, R687-M750, H688-M750, A689-M750,R690-M750, G691-M750, E692-M750, A693-M750, E694-M750, L695-M750,T696-M750, F697-M750, V698-M750, P699-M750, Q700-M750, K701-M750,S702-M750, K703-M750, S704-M750, K705-M750, S706-M750, T707-M750,K708-M750, L709-M750, K710-M750, F711-M750, N712-M750, N713-M750,N714-M750, H715-M750, S716-M750, D717-M750, D718-M750, E719-M750,K720-M750, L721-M750, D722-M750, S723-M750, G724-M750, K725-M750,T726-M750, K727-M750, D728-M750, S729-M750, G730-M750, R731-M750,T732-M750, K733-M750, Q734-M750, R735-M750, F736-M750, E737-M750,G738-M750, R739-M750, R740-M750, I741-M750, A742-M750, S743-M750, and/orK744-M750 of SEQ ID NO:17. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these N-terminal CaYGR145w deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYGR145w deletionpolypeptides are encompassed by the present invention: M1-M750, M1-G749,M1-R748, M1-F747, M1-K746, M1-N745, M1-K744, M1-S743, M1-A742, M1-I741,M1-R740, M1-R739, M1-G738, M1-E737, M1-F736, M1-R735, M1-Q734, M1-K733,M1-T732, M1-R731, M1-G730, M1-S729, M1-D728, M1-K727, M1-T726, M1-K725,M1-G724, M1-S723, M1-D722, M1-L721, M1-K720, M1-E719, M1-D718, M1-D717,M1-S716, M1-H715, M1-N714, M1-N713, M1-N712, M1-F711, M1-K710, M1-L709,M1-K708, M1-T707, M1-S706, M1-K705, M1-S704, M1-K703, M1-S702, M1-K701,M1-Q700, M1-P699, M1-V698, M1-F697, M1-T696, M1-L695, M1-E694, M1-A693,M1-E692, M1-G691, M1-R690, M1-A689, M1-H688, M1-R687, M1-R686, M1-L685,M1-R684, M1-A683, M1-D682, M1-K681, M1-A680, M1-N679, M1-S678, M1-I677,M1-S676, M1-N675, M1-N674, M1-N673, 1-K672, M1-N671, M1-Q670, M1-K669,M1-S668, M1-I667, M1-K666, M1-N665, M1-L664, M1-K663, M1-N662, M1-V661,M1-Q660, M1-S659, M1-G658, M1-F657, M1-S656, M1-V655, M1-S654, M1-A653,M1-K652, M1-E651, M1-T650, M1-N649, M1-A648, M1-L647, M1-S646, M1-S645,M1-S644, M1-S643, M1-S642, M1-S641, M1-S640, M1-Q639, M1-Q638, M1-S637,M1-L636, M1-I635, M1-K634, M1-M633, M1-E632, M1-N631, M1-Q630, M1-F629,M1-K628, M1-K627, M1-S626, M1-D625, M1-Q624, M1-Q623, M1-Q622, M1-Q621,M1-K620, M1-Q619, M1-K618, M1-S617, M1-Q616, M1-R615, M1-I614, M1-K613,M1-N612, M1-L611, M1-E610, M1-K609, M1-G608, M1-V607, M1-R606, M1-E605,M1-R604, M1-T603, M1-E602, M1-A601, M1-S600, M1-K599, M1-A598, M1-E597,M1-D596, M1-E595, M1-S594, M1-Q593, M1-E592, M1-E591, M1-S590, M1-E589,M1-S588, M1-D587, M1-S586, M1-E585, M1-E584, M1-D583, M1-S582, M1-E581,M1-D580, M1-S579, M1-D578, M1-L577, M1-G576, M1-T575, M1-H574, M1-H573,M1-S572, M1-D571, M1-K570, M1-M569, M1-N568, M1-L567, M1-R566, M1-E565,M1-E564, M1-D563, M1-S562, M1-E561, M1-E560, M1-A559, M1-A558, M1-T557,M1-L556, M1-G555, M1-R554, M1-G553, M1-R552, M1-S551, M1-N550, M1-T549,M1-T548, M1-G547, M1-T546, M1-N545, M1-T544, M1-T543, M1-T542, M1-I541,M1-D540, M1-K539, M1-T538, M1-S537, M1-K536, M1-V535, M1-P534, M1-N533,M1-L532, M1-Q531, M1-K530, M1-Y529, M1-E528, M1-H527, M1-S526, M1-E525,M1-E524, M1-D523, M1-I522, M1-E521, M1-F520, M1-D519, M1-P518, M1-N517,M1-E516, M1-F515, M1-M514, M1-E513, M1-R512, M1-F511, M1-R510, M1-D509,M1-D508, M1-N507, M1-V506, M1-I505, M1-S504, M1-E503, M1-V502, M1-Y501,M1-D500, M1-T499, M1-A498, M1-G497, M1-N496, M1-A495, M1-N494, M1-G493,M1-D492, M1-P491, M1-T490, M1-G489, M1-N488, M1-E487, M1-A486, M1-F485,M1-K484, M1-T483, M1-Q482, M1-L481, M1-K480, M1-N479, M1-V478, M1-L477,M1-D476, M1-K475, M1-N474, M1-V473, M1-K472, M1-I471, M1-K470, M1-T469,M1-P468, M1-L467, M1-D466, M1-N465, M1-G464, M1-N463, M1-S462, M1-S461,M1-T460, M1-L459, M1-I458, M1-R457, M1-S456, M1-K455, M1-R454, M1-E453,M1-E452, M1-N451, M1-I450, M1-K449, M1-N448, M1-A447, M1-I446, M1-E445,M1-R444, M1-K443, M1-R442, M1-Q441, M1-D440, M1-Y439, M1-I438, M1-S437,M1-N436, M1-P435, M1-N434, M1-S433, M1-I432, M1-L431, M1-N430, M1-V429,M1-K428, M1-D427, M1-Y426, M1-L425, M1-E424, M1-T423, M1-N422, M1-I421,M1-F420, M1-F419, M1-G418, M1-H417, M1-M416, M1-Y415, M1-S414, M1-R413,M1-I412, M1-V411, M1-K410, M1-I409, M1-G408, M1-I407, M1-L406, M1-H405,M1-T404, M1-L403, M1-N402, M1-L401, M1-K400, M1-V399, M1-M398, M1-E397,M1-D396, M1-R395, M1-T394, M1-I393, M1-F392, M1-K391, M1-F390, M1-T389,M1-P388, M1-Y387, M1-I386, M1-S385, M1-N384, M1-S383, M1-P382, M1-K381,M1-E380, M1-E379, M1-L378, M1-E377, M1-E376, M1-T375, M1-V374, M1-N373,M1-D372, M1-L371, M1-F370, M1-S369, M1-C368, M1-W367, M1-N366, M1-P365,M1-A364, M1-S363, M1-G362, M1-L361, M1-N360, M1-P359, M1-I358, M1-Y357,M1-Y356, M1-T355, M1-H354, M1-M353, M1-P352, M1-M351, M1-G350, M1-E349,M1-N348, M1-A347, M1-M346, M1-F345, M1-F344, M1-M343, M1-G342, M1-S341,M1-Q340, M1-P339, M1-I338, M1-H337, M1-C336, M1-I335, M1-D334, M1-N333,M1-I332, M1-D331, M1-V330, M1-T329, M1-P328, M1-E327, M1-M326, M1-S325,M1-A324, M1-F323, M1-S322, M1-K321, M1-G320, M1-N319, M1-T318, M1-H317,M1-D316, M1-W315, M1-I314, M1-K313, M1-V312, M1-I311, M1-R310, M1-K309,M1-D308, M1-S307, M1-T306, M1-L305, M1-I304, M1-M303, M1-E302, M1-P301,M1-K300, M1-L299, M1-S298, M1-D297, M1-Q296, M1-C295, M1-W294, M1-I293,M1-I292, M1-K291, M1-K290, M1-I289, M1-D288, M1-Y287, M1-G286, M1-Y285,M1-G284, M1-Q283, M1-D282, M1-K281, M1-I280, M1-Q279, M1-Y278, M1-P277,M1-E276, M1-S275, M1-A274, M1-R273, M1-L272, M1-D271, M1-Y270, M1-L269,M1-L268, M1-T267, M1-Q266, M1-G265, M1-N264, M1-S263, M1-T262, M1-G261,M1-C260, M1-A259, M1-F258, M1-N257, M1-L256, M1-A255, M1-D254, M1-Q253,M1-P252, M1-R251, M1-F250, M1-A249, M1-L248, M1-S247, M1-T246, M1-I245,M1-G244, M1-C243, M1-S242, M1-S241, M1-Q240, M1-E239, M1-T238, M1-N237,M1-N236, M1-T235, M1-S234, M1-N233, M1-I232, M1-L231, M1-Q230, M1-D229,M1-S228, M1-V227, M1-F226, M1-L225, M1-K224, M1-G223, M1-A222, M1-R221,M1-Q220, M1-K219, M1-S218, M1-R217, M1-P216, M1-D215, M1-W214, M1-F213,M1-E212, M1-V211, M1-T210, M1-G209, M1-D208, M1-D207, M1-L206, M1-G205,M1-A204, M1-S203, M1-I202, M1-L201, M1-G200, M1-H199, M1-M198, M1-S197,M1-N196, M1-I195, M1-D194, M1-V193, M1-A192, M1-N191, M1-C190, M1-G189,M1-S188, M1-D187, M1-I186, M1-N185, M1-G184, M1-G183, M1-D182, M1-T181,M1-M180, M1-D179, M1-L178, M1-K177, M1-L176, M1-P175, M1-S174, M1-L173,M1-F172, M1-R171, M1-G170, M1-Q169, M1-D168, M1-L167, M1-N166, M1-L165,M1-R164, M1-Y163, M1-L162, M1-E161, M1-D160, M1-S159, M1-S158, M1-S157,M1-G156, M1-V155, M1-I154, M1-L153, M1-D152, M1-C151, M1-N150, M1-I149,M1-P148, M1-N147, M1-Y146, M1-T145, M1-L144, M1-C143, M1-R142, M1-G141,M1-F140, M1-K139, M1-P138, M1-I137, M1-R136, M1-T135, M1-R134, M1-Y133,M1-H132, M1-V131, M1-G130, M1-G129, M1-A128, M1-T127, M1-Q126, M1-F125,M1-E124, M1-I123, M1-S122, M1-R121, M1-D120, M1-C119, M1-Q118, M1-L117,M1-H116, M1-V115, M1-S114, M1-K113, M1-T112, M1-W111, M1-D110, M1-N109,M1-S108, M1-L107, M1-I106, M1-L105, M1-F104, M1-D103, M1-I102, M1-N101,M1-E100, M1-V99, M1-N98, M1-T97, M1-H96, M1-R95, M1-D94, M1-F93, M1-K92,M1-L91, M1-S90, M1-L89, M1-N88, M1-A87, M1-F86, M1-E85, M1-Y84, M1-V83,M1-H82, M1-I81, M1-Q80, M1-P79, M1-K78, M1-Y77, M1-T76, M1-G75, M1-T74,M1-A73, M1-M72, M1-C71, M1-Y70, M1-Q69, M1-G68, M1-D67, M1-N66, M1-T65,M1-V64, M1-K63, M1-I62, M1-K61, M1-N60, M1-S59, M1-A58, M1-E57, M1-S56,M1-F55, M1-E54, M1-F53, M1-D52, M1-Q51, M1-I50, M1-L49, M1-E48, M1-I47,M1-R46, M1-N45, M1-Q44, M1-Y43, M1-E42, M1-L41, M1-D40, M1-H39, M1-K38,M1-L37, M1-A36, M1-R35, M1-K34, M1-R33, M1-K32, M1-K31, M1-D30, M1-I29,M1-W28, M1-D27, M1-P26, M1-L25, M1-S24, M1-R23, M1-S22, M1-V21, M1-N20,M1-T19, M1-G18, M1-S17, M1-V16, M1-Q15, M1-Y14, M1-V13, M1-S12, M1-V11,M1-N10, M1-G9, M1-A8, and/or M1-T7 of SEQ ID NO:17. Polynucleotidesequences encoding these polypeptides are also provided. The presentinvention also encompasses the use of these C-terminal CaYGR145wdeletion polypeptides as immunogenic and/or antigenic epitopes asdescribed elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9% identical to a the polynucleotide sequence provided in SEQ IDNO:6, and in particular to the coding region of the polynucleotidesequence provided in SEQ ID NO:6. Preferably such polynucleotides encodepolypeptides that have biological activity.

The present invention also encompasses polypeptides sharing at leastleast about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9% identical to a the polypeptide sequence provided in SEQ IDNO:17.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:6 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 2236 ofSEQ ID NO:6, b is an integer between 15 to 2250, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:6,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:7

The polynucleotide sequence (SEQ ID NO:7) and deduced amino acidsequence (SEQ ID NO:18) of the novel fungal essential gene, CaYDR412w(also referred to as FCG13), of the present invention. The CaYDR412wpolypeptide (SEQ ID NO:18) is encoded by nucleotides 1 to 804 of SEQ IDNO:7 and has a predicted molecular weight of 31.3 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYDR412w. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 2250 of SEQ ID NO:7, and the polypeptide corresponding to aminoacids 2 thru 268 of SEQ ID NO:18. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYDR412w polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYDR412w deletionpolypeptides are encompassed by the present invention: M1-K268, A2-K268,G3-K268, F4-K268, K5-K268, K6-K268, N7-K268, R8-K268, E9-K268, I10-K268,L111-K268, T12-K268, G13-K268, G14-K268, K15-K268, K16-K268, Y17-K268,I18-K268, Q19-K268, Q20-K268, K21-K268, Q22-K268, K23-K268, K24-K268,H25-K268, L26-K268, V27-K268, D28-K268, E29-K268, V30-K268, V31-K268,F32-K268, D33-K268, K34-K268, E35-K268, S36-K268, R37-K268, H38-K268,E39-K268, Y40-K268, L41-K268, T42-K268, G43-K268, F44-K268, H45-K268,K46-K268, R47-K268, K48-K268, L49-K268, Q50-K268, R51-K268, Q52-K268,K53-K268, K54-K268, A55-K268, Q56-K268, E57-K268, F58-K268, H59-K268,K60-K268, E61-K268, Q62-K268, E63-K268, R64-K268, L65-K268, A66-K268,K67-K268, I68-K268, E69-K268, E70-K268, R71-K268, K72-K268, Q73-K268,L74-K268, K75-K268, Q76-K268, E77-K268, R78-K268, E79-K268, R80-K268,D81-K268, L82-K268, Q83-K268, N84-K268, Q85-K268, L86-K268, Q87-K268,Q88-K268, F89-K268, K90-K268, K91-K268, T92-K268, A93-K268, Q94-K268,E95-K268, I96-K268, A97-K268, A98-K268, I99-K268, N100-K268, N101-K268,D102-K268, I103-K268, G104-K268, F105-K268, D106-K268, Q107-K268,S108-K268, D109-K268, D110-K268, N111-K268, N112-K268, D113-K268,N114-K268, D115-K268, N116-K268, E117-K268, N118-K268, E119-K268,E120-K268, W121-K268, S122-K268, G123-K268, F124-K268, Q125-K268,E126-K268, D127-K268, E128-K268, E129-K268, G130-K268, E131-K268,G132-K268, E133-K268, E134-K268, V135-K268, T136-K268, D137-K268,E138-K268, D139-K268, D140-K268, E141-K268, D142-K268, K143-K268,E144-K268, K145-K268, P146-K268, L147-K268, K148-K268, G149-K268,I150-K268, L151-K268, H152-K268, H153-K268, T154-K268, E155-K268,I156-K268, Y157-K268, K158-K268, Q159-K268, D160-K268, P161-K268,S162-K268, L163-K268, S164-K268, N165-K268, I166-K268, T167-K268,N168-K268, N169-K268, G170-K268, A171-K268, I172-K268, I173-K268,D174-K268, D175-K268, E176-K268, T177-K268, T178-K268, V179-K268,V180-K268, V181-K268, E182-K268, S183-K268, L184-K268, D185-K268,N186-K268, P187-K268, N188-K268, A189-K268, V190-K268, D191-K268,T192-K268, E193-K268, E194-K268, K195-K268, L196-K268, Q197-K268,Q198-K268, L199-K268, A200-K268, K201-K268, L202-K268, N203-K268,N204-K268, V205-K268, N206-K268, L207-K268, D208-K268, K209-K268,S210-K268, D211-K268, Q212-K268, I213-K268, L214-K268, E215-K268,K216-K268, S217-K268, I218-K268, E219-K268, R220-K268, A221-K268,K222-K268, N223-K268, Y224-K268, A225-K268, V226-K268, I227-K268,C228-K268, G229-K268, V230-K268, A231-K268, K232-K268, P233-K268,N234-K268, P235-K268, I236-K268, K237-K268, Q238-K268, K239-K268,K240-K268, K241-K268, K242-K268, F243-K268, R244-K268, Y245-K268,L246-K268, T247-K268, K248-K268, A249-K268, E250-K268, R251-K268,R252-K268, E253-K268, N254-K268, V255-K268, R256-K268, K257-K268,E258-K268, K259-K268, S260-K268, K261-K268, and/or S262-K268 of SEQ IDNO:18. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseN-terminal CaYDR412w deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYDR412w deletionpolypeptides are encompassed by the present invention: M1-K268, M1-K267,M1-G266, M1-K265, M1-S264, M1-K263, M1-S262, M1-K261, M1-S260, M1-K259,M1-E258, M1-K257, M1-R256, M1-V255, M1-N254, M1-E253, M1-R252, M1-R251,M1-E250, M1-A249, M1-K248, M1-T247, M1-L246, M1-Y245, M1-R244, M1-F243,M1-K242, M1-K241, M1-K240, M1-K239, M1-Q238, M1-K237, M1-I236, M1-P235,M1-N234, M1-P233, M1-K232, M1-A231, M1-V230, M1-G229, M1-C228, M1-I227,M1-V226, M1-A225, M1-Y224, M1-N223, M1-K222, M1-A221, M1-R220, M1-E219,M1-I218, M1-S217, M1-K216, M1-E215, M1-L214, M1-1213, M1-Q212, M1-D211,M1-S210, M1-K209, M1-D208, M1-L207, M1-N206, M1-V205, M1-N204, M1-N203,M1-L202, M1-K201, M1-A200, M1-L199, M1-Q198, M1-Q197, M1-L196, M1-K195,M1-E194, M1-E193, M1-T192, M1-D191, M1-V190, M1-A189, M1-N188, M1-P187,M1-N186, M1-D185, M1-L184, M1-S183, M1-E182, M1-V181, M1-V180, M1-V179,M1-T178, M1-T177, M1-E176, M1-D175, M1-D174, M1-I173, M1-I172, M1-A171,M1-G170, M1-N169, M1-N168, M1-T167, M1-I166, M1-N165, M1-S164, M1-L163,M1-S162, M1-P161, M1-D160, M1-Q159, M1-K158, M1-Y157, M1-I156, M1-E155,M1-T154, M1-H153, M1-H152, M1-L151, M1-I150, M1-G149, M1-K148, M1-L147,M1-P146, M1-K145, M1-E144, M1-K143, M1-D142, M1-E141, M1-D140, M1-D139,M1-E138, M1-D137, M1-T136, M1-V135, M1-E134, M1-E133, M1-G132, M1-E131,M1-G130, M1-E129, M1-E128, M1-D127, M1-E126, M1-Q125, M1-F124, M1-G123,M1-S122, M1-W121, M1-E120, M1-E119, M1-N118, M1-E117, M1-N116, M1-D115,M1-N114, M1-D113, M1-N112, M1-N111, M1-D110, M1-D109, M1-S108, M1-Q107,M1-D106, M1-F105, M1-G104, M1-I103, M1-D102, M1-N101, M1-N100, M1-I99,M1-A98, M1-A97, M1-I96, M1-E95, M1-Q94, M1-A93, M1-T92, M1-K91, M1-K90,M1-F89, M1-Q88, M1-Q87, M1-L86, M1-Q85, M1-N84, M1-Q83, M1-L82, M1-D81,M1-R80, M1-E79, M1-R78, M1-E77, M1-Q76, M1-K75, M1-L74, M1-Q73, M1-K72,M1-R71, M1-E70, M1-E69, M1-I68, M1-K67, M1-A66, M1-L65, M1-R64, M1-E63,M1-Q62, M1-E61, M1-K60, M1-H59, M1-F58, M1-E57, M1-Q56, M1-A55, M1-K54,M1-K53, M1-Q52, M1-R51, M1-Q50, M1-L49, M1-K48, M1-R47, M1-K46, M1-H45,M1-F44, M1-G43, M1-T42, M1-L41, M1-Y40, M1-E39, M1-H38, M1-R37, M1-S36,M1-E35, M1-K34, M1-D33, M1-F32, M1-V31, M1-V30, M1-E29, M1-D28, M1-V27,M1-L26, M1-H25, M1-K24, M1-K23, M1-Q22, M1-K21, M1-Q20, M1-Q19, M1-I18,M1-Y17, M1-K16, M1-K15, M1-G14, M1-G13, M1-T12, M1-L11, M1-I0, M1-E9,M1-R8, and/or M1-N7 of SEQ ID NO:18. Polynucleotide sequences encodingthese polypeptides are also provided. The present invention alsoencompasses the use of these C-terminal CaYDR412w deletion polypeptidesas immunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polynucleotide sequence provided in SEQ ID NO:7, andin particular to the coding region of the polynucleotide sequenceprovided in SEQ ID NO:7. Preferably such polynucleotides encodepolypeptides that have biological activity.

The present invention also encompasses polypeptides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polypeptide sequence provided in SEQ ID NO:18.

Most preferred are polypeptides that share at least about 99.4% identitywith the polypeptide sequence provided in SEQ ID NO:18.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:7 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 790 ofSEQ ID NO:7, b is an integer between 15 to 804, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:7,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:8

The polynucleotide sequence (SEQ ID NO:8) and deduced amino acidsequence (SEQ ID NO:19) of the novel fungal essential gene, CaYOL010w(also referred to as FCG14), of the present invention. The CaYOL010wpolypeptide (SEQ ID NO:19) is encoded by nucleotides 1 to 1113 of SEQ IDNO:8 and has a predicted molecular weight of 40.6 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYOL010w. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 1113 of SEQ ID NO:8, and the polypeptide corresponding to aminoacids 2 thru 371 of SEQ ID NO:19. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYOL010w polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYOL010w deletionpolypeptides are encompassed by the present invention: M1-A371, S2-A371,S3-A371, V4-A371, A5-A371, S6-A371, K7-A371, K8-A371, I9-A371, 110-A371,T11-A371, F12-A371, E13-A371, G14-A371, H15-A371, R16-A371, N17-A371,F18-A371, R19-A371, L20-A371, R21-A371, L22-A371, V23-A371, L24-A371,A25-A371, T26-A371, L27-A371, S28-A371, G29-A371, K30-A371, P31-A371,132-A371, K33-A371, 134-A371, T35-A371, K36-A371, 137-A371, R38-A371,S39-A371, Q40-A371, D41-A371, L42-A371, N43-A371, P44-A371, G45-A371,L46-A371, K47-A371, D48-A371, H49-A371, E50-A371, V51-A371, S52-A371,F53-A371, L54-A371, R55-A371, L56-A371, L57-A371, E58-A371, A59-A371,V60-A371, T61-A371, N62-A371, G63-A371, S64-A371, H65-A371, 166-A371,E67-A371, 168-A371, S69-A371, Y70-A371, T71-A371, G72-A371, T73-A371,T74-A371, 175-A371, 176-A371, Y77-A371, R78-A371, P79-A371, G80-A371,181-A371, I82-A371, 183-A371, G84-A371, G85-A371, D86-A371, L87-A371,T88-A371, H89-A371, N90-A371, C91-A371, P92-A371, D93-A371, T94-A371,K95-A371, S96-A371, 197-A371, G98-A371, Y99-A371, F100-A371, 1101-A371,E102-A371, P103-A371, M104-A371, L105-A371, M106-A371, F107-A371,P108-A371, L109-A371, F110-A371, S11′-A371, K112-A371, K113-A371,K114-A371, F115-A371, S116-A371, 1117-A371, 1118-A371, F119-A371,K120-A371, G121-A371, L122-A371, T123-A371, N124-A371, 1125-A371,A126-A371, G127-A371, N128-A371, D129-A371, T130-A371, G131-A371,V132-A371, D133-A371, A134-A371, 1135-A371, K136-A371, W137-A371,G138-A371, L139-A371, L140-A371, P141-A371, V142-A371, M143-A371,E144-A371, K145-A371, F146-A371, G147-A371, V148-A371, R149-A371,E150-A371, V151-A371, S152-A371, L153-A371, H154-A371, 1155-A371,L156-A371, K157-A371, R158-A371, G159-A371, S160-A371, A161-A371,P162-A371, L163-A371, G164-A371, G165-A371, G166-A371, E167-A371,V168-A371, H169-A371, L170-A371, L171-A371, C172-A371, S173-A371,S174-A371, L175-A371, 1176-A371, P177-A371, Q178-A371, P179-A371,L180-A371, T181-A371, 1182-A371, H183-A371, A184-A371, L185-A371,D186-A371, 1187-A371, P188-A371, K189-A371, F190-A371, S191-A371,A192-A371, 1193-A371, R194-A371, G195-A371, V196-A371, A197-A371,Y198-A371, C199-A371, T200-A371, R201-A371, V202-A371, S203-A371,P204-A371, S205-A371, 1206-A371, V207-A371, N208-A371, R209-A371,M210-A371, 1211-A371, D212-A371, S213-A371, A214-A371, R215-A371,A216-A371, V217-A371, L218-A371, K219-A371, P220-A371, T221-A371,G222-A371, C223-A371, E224-A371, V225-A371, N226-A371, I227-A371,T228-A371, A229-A371, D230-A371, V231-A371, W232-A371, R233-A371,G234-A371, E235-A371, N236-A371, S237-A371, G238-A371, K239-A371,S240-A371, P241-A371, G242-A371, F243-A371, G244-A371, 1245-A371,T246-A371, L247-A371, V248-A371, A249-A371, E250-A371, L251-A371,K252-A371, R253-A371, G254-A371, W255-A371, R256-A371, 1257-A371,V258-A371, T259-A371, E260-A371, N261-A371, V262-A371, G263-A371,S264-A371, A265-A371, G266-A371, S267-A371, L268-A371, P269-A371,E270-A371, D271-A371, S272-A371, G273-A371, E274-A371, L275-A371,T276-A371, A277-A371, Y278-A371, Q279-A371, L280-A371, L281-A371,E282-A371, E283-A371, 1284-A371, S285-A371, N286-A371, S287-A371,G288-A371, V289-A371, V290-A371, G291-A371, R292-A371, Y293-A371,Q294-A371, L295-A371, P296-A371, L297-A371, A298-A371, L299-A371,V300-A371, Y301-A371, M302-A371, T303-A371, 1304-A371, G305-A371,K306-A371, E307-A371, D308-A371, 1309-A371, G310-A371, R311-A371,L312-A371, K313-A371, L314-A371, Q315-A371, K316-A371, S317-A371,E318-A371, 1319-A371, D320-A371, E321-A371, N322-A371, L323-A371,V324-A371, S325-A371, V326-A371, L327-A371, R328-A371, D329-A371,1330-A371, Q331-A371, E332-A371, V333-A371, F334-A371, G335-A371,T336-A371, E337-A371, A338-A371, F339-A371, F340-A371, K341-A371,D342-A371, D343-A371, A344-A371, E345-A371, E346-A371, L347-A371,D348-A371, S349-A371, D350-A371, D351-A371, K352-A371, F353-A371,M354-A371, T355-A371, V356-A371, S357-A371, 1358-A371, K359-A371,G360-A371, V361-A371, G362-A371, F363-A371, T364-A371, and/or N365-A371of SEQ ID NO:19. Polynucleotide sequences encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese N-terminal CaYOL010w deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYOL010w deletionpolypeptides are encompassed by the present invention: M1-A371, M1-I370,M1-K369, M1-K368, M1-S367, M1-V366, M1-N365, M1-T364, M1-F363, M1-G362,M1-V361, M1-G360, M1-K359, M1-I358, M1-S357, M1-V356, M1-T355, M1-M354,M1-F353, M1-K352, M1-D351, M1-D350, M1-S349, M1-D348, M1-L347, M1-E346,M1-E345, M1-A344, M1-D343, M1-D342, M1-K341, M1-F340, M1-F339, M1-A338,M1-E337, M1-T336, M1-G335, M1-F334, M1-V333, M1-E332, M1-Q331, M1-I330,M1-D329, M1-R328, M1-L327, M1-V326, M1-S325, M1-V324, M1-L323, M1-N322,M1-E321, M1-D320, M1-I319, M1-E318, M1-S317, M1-K316, M1-Q315, M1-L314,M1-K313, M1-L312, M1-R311, M1-G310, M1-I309, M1-D308, M1-E307, M1-K306,M1-G305, M1-I304, M1-T303, M1-M302, M1-Y301, M1-V300, M1-L299, M1-A298,M1-L297, M1-P296, M1-L295, M1-Q294, M1-Y293, M1-R292, M1-G291, M1-V290,M1-V289, M1-G288, M1-S287, M1-N286, M1-S285, M1-I284, M1-E283, M1-E282,M1-L281, M1-L280, M1-Q279, M1-Y278, M1-A277, M1-T276, M1-L275, M1-E274,M1-G273, M1-S272, M1-D271, M1-E270, M1-P269, M1-L268, M1-S267, M1-G266,M1-A265, M1-S264, M1-G263, M1-V262, M1-N261, M1-E260, M1-T259, M1-V258,M1-I257, M1-R256, M1-W255, M1-G254, M1-R253, M1-K252, M1-L251, M1-E250,M1-A249, M1-V248, M1-L247, M1-T246, M1-I245, M1-G244, M1-F243, M1-G242,M1-P241, M1-S240, M1-K239, M1-G238, M1-S237, M1-N236, M1-E235, M1-G234,M1-R233, M1-W232, M1-V231, M1-D230, M1-A229, M1-T228, M1-I227, M1-N226,M1-V225, M1-E224, M1-C223, M1-G222, M1-T221, M1-P220, M1-K219, M1-L218,M1-V217, M1-A216, M1-R215, M1-A214, M1-S213, M1-D212, M1-I211, M1-M210,M1-R209, M1-N208, M1-V207, M1-I206, M1-S205, M1-P204, M1-S203, M1-V202,M1-R201, M1-T200, M1-C199, M1-Y198, M1-A197, M1-V196, M1-G195, M1-R194,M1-I193, M1-A192, M1-S191, M1-F190, M1-K189, M1-P188, M1-I187, M1-D186,M1-L185, M1-A184, M1-H183, M1-I182, M1-T181, M1-L180, M1-P179, M1-Q178,M1-P177, M1-I176, M1-L175, M1-S174, M1-S173, M1-C172, M1-L171, M1-L170,M1-H169, M1-V168, M1-E167, M1-G166, M1-G165, M1-G164, M1-L163, M1-P162,M1-A161, M1-S160, M1-G159, M1-R158, M1-K157, M1-L156, M1-I155, M1-H154,M1-L153, M1-S152, M1-V151, M1-E150, M1-R149, M1-V148, M1-G147, M1-F146,M1-K145, M1-E144, M1-M143, M1-V142, M1-P141, M1-L140, M1-L139, M1-G138,M1-W137, M1-K136, M1-I135, M1-A134, M1-D133, M1-V132, M1-G131, M1-T130,M1-D129, M1-N128, M1-G127, M1-A126, M1-I125, M1-N124, M1-T123, M1-L122,M1-G121, M1-K120, M1-F119, M1-I118, M1-1117, M1-S116, M1-F115, M1-K114,M1-K113, M1-K112, M1-S111, M1-F110, M1-L109, M1-P108, M1-F107, M1-M106,M1-L105, M1-M104, M1-P103, M1-E102, M1-I101, M1-F100, M1-Y99, M1-G98,M1-I97, M1-S96, M1-K95, M1-T94, M1-D93, M1-P92, M1-C91, M1-N90, M1-H89,M1-T88, M1-L87, M1-D86, M1-G85, M1-G84, M1-I83, M1-I82, M1-I81, M1-G80,M1-P79, M1-R78, M1-Y77, M1-I76, M1-I75, M1-T74, M1-T73, M1-G72, M1-T71,M1-Y70, M1-S69, M1-I68, M1-E67, M1-I66, M1-H65, M1-S64, M1-G63, M1-N62,M1-T61, M1-V60, M1-A59, M1-E58, M1-L57, M1-L56, M1-R55, M1-L54, M1-F53,M1-S52, M1-V51, M1-E50, M1-H49, M1-D48, M1-K47, M1-L46, M1-G45, M1-P44,M1-N43, M1-L42, M1-D41, M1-Q40, M1-S39, M1-R38, M1-I37, M1-K36, M1-T35,M1-I34, M1-K33, M1-I32, M1-P31, M1-K30, M1-G29, M1-S28, M1-L27, M1-T26,M1-A25, M1-L24, M1-V23, M1-L22, M1-R21, M1-L20, M1-R19, M1-F18, M1-N17,M1-R16, M1-H15, M1-G14, M1-E13, M1-F12, M1-T11, M1-I10, M1-I9, M1-K8,and/or M1-K7 of SEQ ID NO:19. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal CaYOL010w deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polynucleotide sequence provided in SEQ ID NO:8, andin particular to the coding region of the polynucleotide sequenceprovided in SEQ ID NO:8. Preferably such polynucleotides encodepolypeptides that have biological activity, particularly RNA 3′-terminalphosphate cyclase activity.

Most preferred are polynucleotides that share at least about 99.5%identity with the polynuclelotide sequence provided in SEQ ID NO:8.

The present invention also encompasses polypeptides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polypeptide sequence provided in SEQ ID NO:19.

Most preferred are polypeptides that share at least about 99.0% identitywith the polypeptide sequence provided in SEQ ID NO:19.

The present invention is also directed to a homology model detailing thethree-dimensional structure of the CaYOL010w polypeptide (SEQ ID NO:19)of the present invention.

Protein threading and molecular modeling of CaYOL010w suggest thatCaYOL010w has a three dimensional fold similar to that of the RNA3′-terminal phosphate cyclase from E. coli (Palm et. al., 1999), ProteinData Bank (PDB, Bernstein et. al., 1977 & Berman et. al., 2000) (ProteinData Bank entry 1QMH; Genbank Accession No. gil12644279; SEQ ID NO:253).Based on sequence, structure, motifs and known cyclase signaturesequences, CaYOL010w contains a novel RNA cyclase domain.

The polypeptide CaYOL010w contains a distinct structural domain similarto the RNA cyclase domains that are a family of RNA modifying enzymesconserved in eucarya, bacteria and archaea.

The three dimensional crystallographic structure for Escherichia coli

RNA 3′-terminal phosphate cyclase has been reported and was depositedinto the Protein Data Bank (Palm et. al., 1999, Bernstein et. al., 1977,Berman et. al., 2000).

The structure (Protein Data Bank, PDB entry 1QMH) of RNA 3′-terminalphosphate cyclase Escherichia coli is a novel fold that consists onknown structural elements connected in a unique manner. The structure ofcyclase consists of two structurally distinct domains. The larger domainis composed of the N-terminal 184 amino acids (1-184) and the C-terminal59 amino acids (280-339). The domain contains three repeats of a foldingunit comprising two α-helices and a four-stranded β-sheet. The smallerdomain of the cyclase, residues 185-279, comprises the same 4 strandedsheet covered by two α-helices but the connection topology is different.The active site of the of RNA 3′-terminal phosphate cyclase contains ahistidine (H309) at the active site that was identified by labeling andlysines in the active site have been reported as part of the catalyticmechanism of nucleotidyl transferases including cyclases. The H309 liesat the bottom of a deep cleft surrounded by 5 loops containing conservedresidues (from across the orthologs of cyclases). A RNA 3′-terminalphosphate cyclase signature sequence is found in the N-terminal regionof the larger domain. This motif H/R-G-X-P-G-G-G-X-V (SEQ ID NO:256) issimilar to the glycine rich loops known to contact ATP, GTP and othernucleotides at binding sites in other enzymes. The histidine or arginineresidue is thought to bind to the nucleotide. For the E. coli RNA3′-terminal phosphate cyclase H158 through V168 comprises the cyclasefunctional signature.

This structure-based information and sequence information from novelgenes can be used to identify other protein family members that sharethis same fold.

The CaYOL010w three dimensional model provides for a specificdescription of the distinct domain and functional/active sites in theRNA 3′-terminal phosphate cyclase, CaYOL010w polypeptide.

The structural domain and functional/active sites are defined by atomiccoordinates (Table 10). Based on this data, the inventors have ascribedthe CaYOL010w polypeptide as having RNA cyclase activity(s) and cellularand systemic regulatory function(s).

The invention also relates to in silico screening methods including insilico docking and methods of structure based drug design which utilizethe three dimensional coordinates of CaYOL010w (Table 10). Also providedare methods of identifying modulators of CaYOL010w that includemodulator building or searching utilizing computer programs andalgorithms. In an embodiment of the invention a method is provided fordesigning potential modulators of CaYOL010w comprising any combinationof steps which utilize said three dimensional structure to design orselect potential modulators.

Homology models are useful when there is no experimental informationavailable on the protein of interest. A three dimensional model can beconstructed on the basis of the known structure of a homologous protein(Greer et. al., 1991, Lesk, et. al., 1992, Levitt, 1992, Cardozo, et.al., 1995, Sali, et. al., 1995).

Those of skill in the art will understand that a homology model isconstructed on the basis of first identifying a template, or, protein ofknown structure which is similar to the protein without known structure.This can be accomplished by through pairwise alignment of sequencesusing such programs as FASTA (Pearson, et. al. 1990) and BLAST(Altschul, et. al., 1990). In cases where sequence similarity is high(greater than 30%) these pairwise comparison methods may be adequate.Likewise, multiple sequence alignments or profile-based methods can beused to align a query sequence to an alignment of multiple (structurallyand biochemically) related proteins. When the sequence similarity islow, more advanced techniques are used such as fold recognition (proteinthreading; Hendlich, et. al., 1990, Koppensteiner et. Al. 2000, Sippl &Weitckus 1992, Sippl 1993), where the compatibility of a particularsequence with the three dimensional fold of a potential template proteinis gauged on the basis of a knowledge-based potential. Following theinitial sequence alignment, the query template can be optimally alignedby manual manipulation or by incorporation of other features (motifs,secondary structure predictions, and allowed sequence conservation).Next, structurally conserved regions can be identified and are used toconstruct the core secondary structure (Levitt, 1992, Sali, et. al.,1995) elements in the three dimensional model. Variable regions, called“unconserved regions” and loops can be added using knowledge-basedtechniques. The complete model with variable regions and loops can berefined performing forcefield calculations (Sali, et. al., 1995,Cardozo, et. al., 1995).

For CaYOL010w a pairwise alignment generated by protein threading(Hendlich, et. al., 1990, Koppensteiner et. Al. 2000, Sippl & Weitckus1992, Sippl 1993) was used to align the sequence of CaYOL010w with thesequence the RNA 3′-terminal phosphate cyclase, Escherichia coli (Palmet. al., 1999), (Protein Data Bank code 1QMH). The alignment ofCaYOL010w with PDB entry 1QMH chain A is set forth in FIG. 32. In thisinvention, the homology model of CaYOL010w was derived from the sequencealignment set forth in FIG. 32. An overall atomic model includingplausible sidechain orientations was generated using the program LOOK(Levitt, 1992). The three dimensional model for CaYOL010w is defined bythe set of structure coordinates as set forth in Table 10 and is shownin FIG. 33 rendered by backbone secondary structures.

In order to recognize errors in three-dimensional structures knowledgebased mean fields can be used to judge the quality of protein folds(Sippl 1993). The methods can be used to recognize misfolded structuresas well as faulty parts of structural models. The technique generates anenergy graph where the energy distribution for a given protein fold isdisplayed on the y-axis and residue position in the protein fold isdisplayed on the x-axis. The knowledge based mean fields compose a forcefield derived from a set of globular protein structures taken as asubset from the Protein Data Bank (Bernstein et. al. 1977). To analyzethe quality of a model the energy distribution is plotted and comparedto the energy distribution of the template from which the model wasgenerated. FIG. 34 shows the energy graph for the CaYOL010w model(dotted line) and the template (RNA 3′-terminal phosphate cyclase) fromwhich the model was generated. The model has virtually an identicalenergy plot when compared to RNA 3′-terminal phosphate cyclase templatedemonstrating that CaYOL010w has similar structural characteristics andsuggest the overall three-dimensional fold is “native-like”. This graphsupports the motif and sequence alignments in confirming that the threedimensional structure coordinates of CaYOL010w are an accurate anduseful representation for the polypeptide.

The term “structure coordinates” refers to Cartesian coordinatesgenerated from the building of a homology model.

Those of skill in the art will understand that a set of structurecoordinates for a protein is a relative set of points that define ashape in three dimensions. Thus, it is possible that an entirelydifferent set of coordinates could define a similar or identical shape.Moreover, slight variations in the individual coordinates, as emanatefrom generation of similar homology models using different alignmenttemplates (i.e., other than the structure coordinates of 1QMH), and/orusing different methods in generating the homology model, will haveminor effects on the overall shape. Variations in coordinates may alsobe generated because of mathematical manipulations of the structurecoordinates. For example, the structure coordinates set forth in Table10 could be manipulated by fractionalization of the structurecoordinates; integer additions or subtractions to sets of the structurecoordinates, inversion of the structure coordinates or any combinationof the above.

Various computational analyses are therefore necessary to determinewhether a molecule or a portion thereof is sufficiently similar to allor parts of CaYOL010w described above as to be considered the same. Suchanalyses may be carried out in current software applications, such asINSIGHTII (Accelrys Inc., San Diego, Calif.) version 2000 as describedin the User's Guide, online or software applications available in theSYBYL software suite (Tripos Inc., St. Louis, Mo.).

Using the superimposition tool in the program INSIGHTII comparisons canbe made between different structures and different conformations of thesame structure. The procedure used in INSIGHTII to compare structures isdivided into four steps: 1) load the structures to be compared; 2)define the atom equivalencies in these structures; 3) perform a fittingoperation; and 4) analyze the results. Each structure is identified by aname. One structure is identified as the target (i.e., the fixedstructure); the second structure (i.e., moving structure) is identifiedas the source structure. Since atom equivalency within INSIGHTII isdefined by user input, for the purpose of this invention we will defineequivalent atoms as protein backbone atoms (N, Cα, C and O) for allconserved residues between the two structures being compared. We willalso consider only rigid fitting operations. When a rigid fitting methodis used, the working structure is translated and rotated to obtain anoptimum fit with the target structure. The fitting operation uses analgorithm that computes the optimum translation and rotation to beapplied to the moving structure, such that the root mean squaredifference of the fit over the specified pairs of equivalent atom is anabsolute minimum. This number, given in angstroms, is reported byINSIGHTII.

For the purpose of this invention, any homology model of a CaYOL010wthat has a root mean square deviation of conserved residue backboneatoms (N, Cα, C, O) of less than 2.0 A when superimposed on the relevantbackbone atoms described by structure coordinates listed in Table 10 areconsidered identical. More preferably, the root mean square deviation isless than 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 Angstroms.

The term “root mean square deviation” means the square root of thearithmetic mean of the squares of the deviations from the mean. It is away to express the deviation or variation from a trend or object. Forpurposes of this invention, the “root mean square deviation” defines thevariation in the backbone of a protein from the relevant portion of thebackbone of CaYOL010w as defined by the structure coordinates describedherein.

This invention as embodied by the three-dimensional model enables thestructure-based design of modulators of the biological function ofCaYOL010w, as well as mutants with altered biological function and/orspecificity.

The sequence alignment (FIG. 32) used as a template for creating thethree-dimensional model of CaYOL010w RNA 3′-terminal phosphate cyclasedomain shows 24% sequence identity between catalytic domain of CaYOL010wand yeast RNA 3′-terminal phosphate cyclase, PDB code 1QMH. For the RNA3′-terminal phosphate cyclases there are at least two functional regionsthat are critical. In the N-terminal region of the enzyme the cyclasesignature motif is thought to contain the nucleotide binding site hasbeen shown to be highly conserved among cyclases. FIG. 32 shows thisregion highlighted by (*) and corresponds to R158-V168 in the threedimensional model for YOL010w (Table 10). The nucleotide binding siteand surrounding sequence is completely conserved at the sequence andstructure level. The second functional site is the region correspondingto RNA 3′-terminal phosphate cyclase H309. FIG. 32 shows that thisregion is not conserved in the model but there are several lysines andarginines nearby in the sequence alignment that suggest that thisprotein utilizes a basic residue like the DNA or RNA ligases. Thenucleotidyl group is transferred and a covalent lysyl-NMP intermediateis formed. The conservation of the amino acids in the functional sitesand the overall 24% sequence identity emphasize the significance of thethree-dimensional model. The conserved residues are located in thefunctional sites at the cyclase signature sequence which is the presumednucleotide binding site and region of the cyclase active site presentinga well structured catalytic domain. These functional site residues playcritical roles in the mechanism of catalysis, substrate specificity andRNA processing and modification.

The structure coordinates of a CaYOL010w homology model, and portionsthereof, are stored in a machine-readable storage medium. Such data maybe used for a variety of purposes, such as drug discovery and targetprioritization and validation.

Accordingly, in one embodiment of this invention is provided amachine-readable data storage medium comprising a data storage materialencoded with the structure coordinates set forth in Table 10.

For the first time, the present invention permits the use, throughhomology modeling based upon the sequence of CaYOL010w (FIGS. 18 and 33)of structure-based or rational drug design techniques to design, select,and synthesizes chemical entities that are capable of modulating thebiological function of CaYOL010w. Comparison of the CaYOL010w homologymodel with the structures of other the RNA cyclases enable the use ofrational or structure based drug design methods to design, select orsynthesize specific chemical modulators of CaYOL010w.

Accordingly, the present invention is also directed to the entiresequence in FIG. 18 or any portion thereof for the purpose of generatinga homology model for the purpose of three dimensional structure-baseddrug designs.

For purposes of this invention, we include mutants or homologues of thesequence in FIG. 18 or any portion thereof. In a preferred embodiment,the mutants or homologues have at least 25% identity, more preferably50% identity, more preferably 75% identity, and most preferably 90%identity to the amino acid residues in FIG. 18.

The three-dimensional model structure of the CaYOL010w will also providemethods for identifying modulators of biological function. Variousmethods or combination thereof can be used to identify these compounds.

Structure coordinates of the active site region defined above can alsobe used to identify structural and chemical features. Identifiedstructural or chemical features can then be employed to design or selectcompounds as potential CaYOL010w modulators. By structural and chemicalfeatures it is meant to include, but is not limited to, van der Waalsinteractions, hydrogen bonding interactions, charge interaction,hydrophobic interactions, and dipole interaction. Alternatively, or inconjunction, the three-dimensional structural model can be employed todesign or select compounds as potential CaYOL010w modulators. Compoundsidentified as potential CaYOL010w modulators can then be synthesized andscreened in an assay characterized by binding of a test compound to theCaYOL010w, or in characterizing CaYOL010w deactivation in the presenceof a small molecule. Examples of assays useful in screening of potentialCaYOL010w modulators include, but are not limited to, screening insilico, in vitro assays and high throughput assays. Finally, thesemethods may also involve modifying or replacing one or more amino acidsfrom CaYOL010w according to Table 10.

However, as will be understood by those of skill in the art upon thisdisclosure, other structure based design methods can be used. Variouscomputational structure based design methods have been disclosed in theart.

For example, a number of computer modeling systems are available inwhich the sequence of the CaYOL010w and the CaYOL010w structure (i.e.,atomic coordinates of CaYOL010w and/or the atomic coordinates of theactive site region as provided in Table 10) can be input. The computersystem then generates the structural details of one or more theseregions in which a potential CaYOL010w modulator binds so thatcomplementary structural details of the potential modulators can bedetermined. Design in these modeling systems is generally based upon thecompound being capable of physically and structurally associating withCaYOL010w. In addition, the compound must be able to assume aconformation that allows it to associate with CaYOL010w. Some modelingsystems estimate the potential inhibitory or binding effect of apotential CaYOL010w modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their abilityto associate with a given protein target are well known. Often thesemethods begin by visual inspection of the binding site on the computerscreen. Selected fragments or chemical entities are then positioned inone or more positions and orientations within the active site region inCaYOL010w. Molecular docking is accomplished using software such asINSIGHTII, ICM (Molsoft LLC, La Jolla, Calif.), and SYBYL, following byenergy minimization and molecular dynamics with standard molecularmechanic forcefields such as CHARMM and MMFF. Examples of computerprograms which assist in the selection of chemical fragment or chemicalentities useful in the present invention include, but are not limitedto, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntzet. al. 1982).

Alternatively, compounds may be designed de novo using either an emptyactive site or optionally including some portion of a known inhibitor.Methods of this type of design include, but are not limited to LUDI(Bohm 1992), LeapFrog (Tripos Associates, St. Louis Mo.) and DOCK (Kuntzet. al., 1982). Programs such as DOCK (Kuntz et. al. 1982) can be usedwith the atomic coordinates from the homology model to identifypotential ligands from databases or virtual databases which potentiallybind the in the active site region, and which may therefore be suitablecandidates for synthesis and testing. The computer programs may utilizea combination of the following steps:

1) Selection of fragments or chemical entities from a database and thenpositioning the chemical entity in one or more orientations within theCaYOL010w catalytic domain defined by Table 10.

2) Characterization of the structural and chemical features of thechemical entity and active site including van der Waals interactions,hydrogen bonding interactions, charge interaction, hydrophobic bondinginteraction, and dipole interactions

3) Search databases for molecular fragments which can be joined to orreplace the docked chemical entity and spatially fit into regionsdefined by the said CaYOL010w catalytic domain or catalytic domainfunctional sites

4) Evaluate the docked chemical entity and fragments using a combinationof scoring schemes which account for van der Waals interactions,hydrogen bonding interactions, charge interaction, hydrophobicinteractions

Databases that may be used include ACD (Molecular Designs Limited),Aldrich (Aldrich Chemical Company), NCI (National Cancer Institute),Maybridge (Maybridge Chemical Company Ltd), CCDC (CambridgeCrystallographic Data Center), CAST (Chemical Abstract Service), Derwent(Derwent Information Limited).

Upon selection of preferred chemical entities or fragments, theirrelationship to each other and CaYOL010w can be visualized and thenassembled into a single potential modulator. Programs useful inassembling the individual chemical entities include, but are not limitedto SYBYL and LeapFrog (Tripos Associates, St. Louis Mo.), LUDI (Bohm1992) as well as 3D Database systems (Martin 1992).

Additionally, the three-dimensional homology model of CaYOL010w will aidin the design of mutants with altered biological activity. Site directedmutagenesis can be used to generate proteins with similar or varyingdegrees of biological activity compared to native CaYOL010w. Thisinvention also relates to the generation of mutants or homologs ofCaYOL010w. It is clear that molecular modeling using the threedimensional structure coordinates set forth in Table 10 andvisualization of the CaYOL010w model, FIG. 34 can be utilized to designhomologs or mutant polypeptides of CaYOL010w that have similar oraltered biological activities, function or reactivities.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:8 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 1099 ofSEQ ID NO:8, b is an integer between 15 to 1113, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:8,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:9

The polynucleotide sequence (SEQ ID NO:9) and deduced amino acidsequence (SEQ ID NO:20) of the novel fungal essential gene, CaYOR004w(also referred to as FCG15), of the present invention. The CaYOR004wpolypeptide (SEQ ID NO:20) is encoded by nucleotides 1 to 771 of SEQ IDNO:9 and has a predicted molecular weight of 29.5 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYOR004w. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 771 of SEQ ID NO:9, and the polypeptide corresponding to aminoacids 2 thru 257 of SEQ ID NO:20. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYOR004w polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYOR004w deletionpolypeptides are encompassed by the present invention: M1-N257, R2-N257,Q3-N257, K4-N257, R5-N257, A6-N257, K7-N257, A8-N257, Y9-N257, K10-N257,K11-N257, Q12-N257, M13-N257, S14-N257, V15-N257, Y16-N257, V17-N257,H18-N257, A19-N257, F20-N257, K21-N257, F22-N257, R23-N257, E24-N257,P25-N257, Y26-N257, Q27-N257, I28-N257, I29-N257, V30-N257, D31-N257,N32-N257, E33-N257, L34-N257, I35-N257, T36-N257, T37-N257, C38-N257,Q39-N257, S40-N257, A41-N257, S42-N257, F43-N257, D44-N257, I45-N257,N46-N257, K47-N257, G48-N257, F49-N257, T50-N257, R51-N257, T52-N257,I53-N257, Q54-N257, A55-N257, E56-N257, N57-N257, K58-N257, P59-N257,M60-N257, I61-N257, T62-N257, Q63-N257, C64-N257, C65-N257, I66-N257,Q67-N257, A68-N257, L69-N257, Y70-N257, D71-N257, T72-N257, K73-N257,N74-N257, Q75-N257, P76-N257, A77-N257, I78-N257, D79-N257, I80-N257,A81-N257, K82-N257, S83-N257, F84-N257, E85-N257, R86-N257, R87-N257,K88-N257, C89-N257, N90-N257, H91-N257, R92-N257, E93-N257, A94-N257,I95-N257, D96-N257, P97-N257, S98-N257, Q99-N257, C100-N257, I101-N257,E102-N257, S103-N257, I104-N257, V105-N257, N106-N257, I107-N257,K108-N257, G109-N257, Q110-N257, N111-N257, K112-N257, H113-N257,R114-N257, Y115-N257, I116-N257, V117-N257, A118-N257, S119-N257,Q120-N257, D121-N257, L122-N257, Q123-N257, L124-N257, R125-N257,K126-N257, K127-N257, L128-N257, R129-N257, K130-N257, I131-N257,P132-N257, G133-N257, V134-N257, P135-N257, L136-N257, I137-N257,Y138-N257, M139-N257, N140-N257, R141-N257, S142-N257, V143-N257,M144-N257, V145-N257, M146-N257, E147-N257, P148-N257, I149-N257,S150-N257, D151-N257, V152-N257, S153-N257, N154-N257, Q155-N257,Y156-N257, N157-N257, M158-N257, N159-N257, Y160-N257, E161-N257,S162-N257, K163-N257, K164-N257, L165-N257, T166-N257, G167-N257,G168-N257, L169-N257, N170-N257, D171-N257, I172-N257, E173-N257,A174-N257, G175-N257, K176-N257, L177-N257, E178-N257, K179-N257,Q180-N257, N181-N257, E182-N257, G183-N257, E184-N257, D185-N257,G186-N257, D187-N257, G188-N257, D189-N257, E190-N257, L191-N257,E192-N257, V193-N257, K194-N257, K195-N257, K196-N257, K197-N257,R198-N257, K199-N257, G200-N257, P201-N257, K202-N257, E203-N257,P204-N257, N205-N257, P206-N257, L207-N257, S208-N257, V209-N257,K210-N257, K211-N257, K212-N257, K213-N257, T214-N257, D215-N257,N216-N257, A217-N257, T218-N257, A219-N257, A220-N257, S221-N257,T222-N257, N223-N257, Q224-N257, E225-N257, Q226-N257, K227-N257,K228-N257, K229-N257, P230-N257, N231-N257, R232-N257, R233-N257,K234-N257, R235-N257, H236-N257, A237-N257, Q238-N257, V239-N257,K240-N257, S241-N257, R242-N257, R243-N257, E244-N257, G245-N257,R246-N257, P247-N257, R248-N257, T249-N257, G250-N257, and/or A251-N257of SEQ ID NO:20. Polynucleotide sequences encoding these polypeptidesare also provided. The present invention also encompasses the use ofthese N-terminal CaYOR004w deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYOR004w deletionpolypeptides are encompassed by the present invention: M1-N257, M1-N256,M1-S255, M1-R254, M1-E253, M1-S252, M1-A251, M1-G250, M1-T249, M1-R248,M1-P247, M1-R246, M1-G245, M1-E244, M1-R243, M1-R242, M1-S241, M1-K240,M1-V239, M1-Q238, M1-A237, M1-H236, M1-R235, M1-K234, M1-R233, M1-R232,M1-N231, M1-P230, M1-K229, M1-K228, M1-K227, M1-Q226, M1-E225, M1-Q224,M1-N223, M1-T222, M1-S221, M1-A220, M1-A219, M1-T218, M1-A217, M1-N216,M1-D215, M1-T214, M1-K213, M1-K212, M1-K211, M1-K210, M1-V209, M1-S208,M1-L207, M1-P206, M1-N205, M1-P204, M1-E203, M1-K202, M1-P201, M1-G200,M1-K199, M1-R198, M1-K197, M1-K196, M1-K195, M1-K194, M1-V193, M1-E192,M1-L191, M1-E190, M1-D189, M1-G188, M1-D187, M1-G186, M1-D185, M1-E184,M1-G183, M1-E182, M1-N181, M1-Q180, M1-K179, M1-E178, M1-L177, M1-K176,M1-G175, M1-A174, M1-E173, M1-I172, M1-D171, M1-N170, M1-L169, M1-G168,M1-G167, M1-T166, M1-L165, M1-K164, M1-K163, M1-S162, M1-E161, M1-Y160,M1-N159, M1-M158, M1-N157, M1-Y156, M1-Q155, M1-N154, M1-S153, M1-V152,M1-D151, M1-S150, M1-I149, M1-P148, M1-E147, M1-M146, M1-V145, M1-M144,M1-V143, M1-S142, M1-R141, M1-N140, M1-M139, M1-Y138, M1-I137, M1-L136,M1-P135, M1-V134, M1-G133, M1-P132, M1-I131, M1-K130, M1-R129, M1-L128,M1-K127, M1-K126, M1-R125, M1-L124, M1-Q123, M1-L122, M1-D121, M1-Q120,M1-S119, M1-A118, M1-V117, M1-I116, M1-Y115, M1-R114, M1-H113, M1-K112,M1-N111, M1-Q110, M1-G109, M1-K108, M1-I107, M1-N106, M1-V105, M1-I104,M1-S103, M1-E102, M1-I101, M1-C100, M1-Q99, M1-S98, M1-P97, M1-D96,M1-I95, M1-A94, M1-E93, M1-R92, M1-H91, M1-N90, M1-C89, M1-K88, M1-R87,M1-R86, M1-E85, M1-F84, M1-S83, M1-K82, M1-A81, M1-I80, M1-D79, M1-I78,M1-A77, M1-P76, M1-Q75, M1-N74, M1-K73, M1-T72, M1-D71, M1-Y70, M1-L69,M1-A68, M I-Q67, M1-I66, M1-C65, M1-C64, M1-Q63, M1-T62, M1-I61, M1-M60,M1-P59, M1-K58, M1-N57, M1-E56, M1-A55, M1-Q54, M1-I53, M1-T52, M1-R51,M1-T50, M1-F49, M1-G48, M1-K47, M1-N46, M1-I45, M1-D44, M1-F43, M1-S42,M1-A41, M1-S40, M1-Q39, M1-C38, M1-T37, M1-T36, M1-I35, M1-L34, M1-E33,M1-N32, M1-D31, M1-V30, M1-I29, M1-I28, M1-Q27, M1-Y26, M1-P25, M1-E24,M1-R23, M1-F22, M1-K21, M1-F20, M1-A19, M1-H18, M1-V17, M1-Y16, M1-V15,M1-S14, M1-M13, M1-Q12, M1-K11, M1-K10, M1-Y9, M1-A8, and/or M1-K7 ofSEQ ID NO:20. Polynucleotide sequences encoding these polypeptides arealso provided. The present invention also encompasses the use of theseC-terminal CaYOR004w deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polynucleotide sequence provided in SEQ ID NO:9, andin particular to the coding region of the polynucleotide sequenceprovided in SEQ ID NO:9. Preferably such polynucleotides encodepolypeptides that have biological activity.

Most preferred are polynucleotides that share at least about 88.9%identity with the polynuclelotide sequence provided in SEQ ID NO:9.

The present invention also encompasses polypeptides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polypeptide sequence provided in SEQ ID NO:20.

Most preferred are polypeptides that share at least about 95.4% identitywith the polypeptide sequence provided in SEQ ID NO:20.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:9 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 757 ofSEQ ID NO:9, b is an integer between 15 to 771, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:9,and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:10

The polynucleotide sequence (SEQ ID NO:10) and deduced amino acidsequence (SEQ ID NO:21) of the novel fungal essential gene, CaYOR056c(also referred to as FCG16), of the present invention. The CaYOR056cpolypeptide (SEQ ID NO:21) is encoded by nucleotides 1 to 1398 of SEQ IDNO:10 and has a predicted molecular weight of 52.6 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYOR056c. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 1398 of SEQ ID NO:10, and the polypeptide corresponding to aminoacids 2 thru 466 of SEQ ID NO:21. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYOR056c polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYOR056c deletionpolypeptides are encompassed by the present invention: M1-K466, S2-K466,E3-K466, T4-K466, K5-K466, N6-K466, I7-K466, E8-K466, S9-K466, L10-K466,I11-K466, S12-K466, D13-K466, A14-K466, G15-K466, P16-K466, L17-K466,I18-K466, T19-K466, Q20-K466, P21-K466, A22-K466, T23-K466, T24-K466,L25-K466, Q26-K466, Q27-K466, Y28-K466, A29-K466, T30-K466, A31-K466,Y32-K466, Y33-K466, T34-K466, T35-K466, P36-K466, G37-K466, V38-K466,H39-K466, S40-K466, E41-K466, I42-K466, K43-K466, D44-K466, E45-K466,Y46-K466, A47-K466, R48-K466, Q49-K466, Q50-K466, L51-K466, A52-K466,I53-K466, W54-K466, G55-K466, D56-K466, S57-K466, L58-K466, K59-K466,I60-K466, K61-K466, Q62-K466, P63-K466, K64-K466, Q65-K466, E66-K466,Y67-K466, I68-K466, D69-K466, R70-K466, V71-K466, V72-K466, K73-K466,F74-K466, A75-K466, K76-K466, L77-K466, T78-K466, G79-K466, D80-K466,Y81-K466, S82-K466, V83-K466, L84-K466, S85-K466, V86-K466, N87-K466,D88-K466, L89-K466, H90-K466, I91-K466, V92-K466, A93-K466, L94-K466,A95-K466, Y96-K466, E97-K466, L98-K466, E99-K466, C100-K466, L101-K466,N102-K466, N103-K466, G104-K466, E105-K466, D106-K466, N107-K466,L108-K466, R109-K466, S110-K466, F111-K466, P112-K466, G113-K466,E114-K466, V115-K466, L116-K466, K117-K466, N118-K466, Q119-K466,Q120-K466, A121-K466, E122-K466, N123-K466, E124-K466, N125-K466,G126-K466, S127-K466, N128-K466, K129-K466, M130-K466, S131-K466,N132-K466, I133-K466, I134-K466, G135-K466, D136-K466, D137-K466,D138-K466, G139-K466, F140-K466, V141-K466, V142-K466, A143-K466,T144-K466, K145-K466, R146-K466, R147-K466, G148-K466, G149-K466,R150-K466, R151-K466, Q152-K466, R153-K466, E154-K466, K155-K466,A156-K466, E157-K466, L158-K466, R159-K466, K160-K466, K161-K466,G162-K466, L163-K466, L164-K466, P165-K466, T166-K466, F167-K466,S168-K466, P169-K466, K170-K466, P171-K466, K172-K466, G173-K466,G174-K466, L175-K466, E176-K466, T177-K466, E178-K466, E179-K466,P180-K466, N181-K466, E182-K466, L183-K466, S184-K466, N185-K466,D186-K466, K187-K466, T188-K466, I189-K466, D190-K466, E191-K466,T192-K466, P193-K466, Q194-K466, T195-K466, D196-K466, L197-K466,I198-K466, K199-K466, G200-K466, V201-K466, D202-K466, V203-K466,Q204-K466, E205-K466, Q206-K466, E207-K466, S208-K466, Q209-K466,E210-K466, E211-K466, P212-K466, V213-K466, S214-K466, E215-K466,S216-K466, N217-K466, T218-K466, V219-K466, G220-K466, L221-K466,D222-K466, E223-K466, I224-K466, T225-K466, E226-K466, E227-K466,Y228-K466, N229-K466, E230-K466, D231-K466, D232-K466, D233-K466,D234-K466, G235-K466, E236-K466, W237-K466, I238-K466, T239-K466,P240-K466, E241-K466, N242-K466, L243-K466, Q244-K466, E245-K466,E246-K466, I247-K466, I248-K466, K249-K466, D250-K466, K251-K466,N252-K466, E253-K466, Q254-K466, V255-K466, Q256-K466, E257-K466,S258-K466, N259-K466, T260-K466, N261-K466, G262-K466, P263-K466,L264-K466, I265-K466, K266-K466, V267-K466, A268-K466, L269-K466,A270-K466, T271-K466, G272-K466, D273-K466, F274-K466, A275-K466,C276-K466, Q277-K466, N278-K466, V279-K466, A280-K466, M281-K466,Q282-K466, I283-K466, G284-K466, I285-K466, K286-K466, L287-K466,L288-K466, N289-K466, A290-K466, M291-K466, S292-K466, G293-K466,K294-K466, Q295-K466, I296-K466, T297-K466, R298-K466, V299-K466,R300-K466, N301-K466, Y302-K466, M303-K466, Y304-K466, R305-K466,C306-K466, H307-K466, A308-K466, C309-K466, F310-K466, R311-K466,L312-K466, T313-K466, P314-K466, M315-K466, S316-K466, K317-K466,D318-K466, G319-K466, R320-K466, P321-K466, K322-K466, H323-K466,F324-K466, C325-K466, P326-K466, K327-K466, C328-K466, G329-K466,G330-K466, N331-K466, T332-K466, L333-K466, L334-K466, R335-K466,C336-K466, A337-K466, V338-K466, S339-K466, V340-K466, D341-K466,N342-K466, K343-K466, T344-K466, G345-K466, K346-K466, I347-K466,T348-K466, P349-K466, H350-K466, L351-K466, K352-K466, Q353-K466,N354-K466, F355-K466, Q356-K466, W357-K466, I358-K466, R359-K466,R360-K466, G361-K466, E362-K466, R363-K466, Y364-K466, S365-K466,L366-K466, P367-K466, S368-K466, P369-K466, L370-K466, S371-K466,K372-K466, N373-K466, Q374-K466, K375-K466, K376-K466, L377-K466,Q378-K466, G379-K466, N380-K466, G381-K466, G382-K466, Y383-K466,Q384-K466, H385-K466, N386-K466, K387-K466, E388-K466, N389-K466,R390-K466, H391-K466, K392-K466, S393-K466, L394-K466, Q395-K466,T396-K466, P397-K466, L398-K466, I399-K466, L400-K466, N401-K466,E402-K466, D403-K466, Q404-K466, K405-K466, E406-K466, Y407-K466,Q408-K466, R409-K466, A410-K466, L411-K466, K412-K466, N413-K466,D414-K466, E415-K466, W416-K466, E417-K466, R418-K466, K419-K466,Q420-K466, Q421-K466, D422-K466, K423-K466, M424-K466, L425-K466,Q426-K466, E427-K466, W428-K466, I429-K466, G430-K466, G431-K466,G432-K466, S433-K466, A434-K466, D435-K466, N436-K466, F437-K466,V438-K466, S439-K466, P440-K466, F441-K466, G442-K466, N443-K466,T444-K466, I445-K466, R446-K466, N447-K466, S448-K466, G449-K466,V450-K466, K451-K466, V452-K466, G453-K466, R454-K466, G455-K466,R456-K466, Y457-K466, A458-K466, N459-K466, and/or S460-K466 of SEQ IDNO:21. Polynucleotide sequences encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseN-terminal CaYOR056c deletion polypeptides as immunogenic and/orantigenic epitopes as described elsewhere herein.

In preferred embodiments, the following C-terminal CaYOR056c deletionpolypeptides are encompassed by the present invention: M1-K466, M1-R465,M1-K464, M1-K463, M1-K462, M1-S461, M1-S460, M1-N459, M1-A458, M1-Y457,M1-R456, M1-G455, M1-R454, M1-G453, M1-V452, M1-K451, M1-V450, M1-G449,M1-S448, M1-N447, M1-R446, M1-I445, M1-T444, M1-N443, M1-G442, M1-F441,M1-P440, M1-S439, M1-V438, M1-F437, M1-N436, M1-D435, M1-A434, M1-S433,M1-G432, M1-G431, M1-G430, M1-I429, M1-W428, M1-E427, M1-Q426, M1-L425,M1-M424, M1-K423, M1-D422, M1-Q421, M1-Q420, M1-K419, M1-R418, M1-E417,M1-W416, M1-E415, M1-D414, M1-N413, M1-K412, M1-L411, M1-A410, M1-R409,M1-Q408, M1-Y407, M1-E406, M1-K405, M1-Q404, M1-D403, M1-E402, M1-N401,M1-L400, M1-I399, M1-L398, M1-P397, M1-T396, M1-Q395, M1-L394, M1-S393,M1-K392, M1-H391, M1-R390, M1-N389, M1-E388, M1-K387, M1-N386, M1-H385,M1-Q384, M1-Y383, M1-G382, M1-G381, M1-N380, M1-G379, M1-Q378, M1-L377,M1-K376, M1-K375, M1-Q374, M1-N373, M1-K372, M1-S371, M1-L370, M1-P369,M1-S368, M1-P367, M1-L366, M1-S365, M1-Y364, M1-R363, M1-E362, M1-G361,M1-R360, M1-R359, M1-I358, M1-W357, M1-Q356, M1-F355, M1-N354, M1-Q353,M1-K352, M1-L351, M1-H350, M1-P349, M1-T348, M1-I347, M1-K346, M1-G345,M1-T344, M1-K343, M1-N342, M1-D341, M1-V340, M1-S339, M1-V338, M1-A337,M1-C336, M1-R335, M1-L334, M1-L333, M1-T332, M1-N331, M1-G330, M1-G329,M1-C328, M1-K327, M1-P326, M1-C325, M1-F324, M1-H323, M1-K322, M1-P321,M1-R320, M1-G319, M1-D318, M1-K317, M1-S316, M1-M315, M1-P314, M1-T313,M1-L312, M1-R311, M1-F310, M1-C309, M1-A308, M1-H307, M1-C306, M1-R305,M1-Y304, M1-M303, M1-Y302, M1-N301, M1-R300, M1-V299, M1-R298, M1-T297,M1-I296, M1-Q295, M1-K294, M1-G293, M1-S292, M1-M291, M1-A290, M1-N289,M1-L288, M1-L287, M1-K286, M1-I285, M1-G284, M1-I283, M1-Q282, M1-M281,M1-A280, M1-V279, M1-N278, M1-Q277, M1-C276, M1-A275, M1-F274, M1-D273,M1-G272, M1-T271, M1-A270, M1-L269, M1-A268, M1-V267, M1-K266, M1-I265,M1-L264, M1-P263, M1-G262, M1-N261, M1-T260, M1-N259, M1-S258, M1-E257,M1-Q256, M1-V255, M1-Q254, M1-E253, M1-N252, M1-K251, M1-D250, M1-K249,M1-I248, M1-I247, M1-E246, M1-E245, M1-Q244, M1-L243, M1-N242, M1-E241,M1-P240, M1-T239, M1-I238, M1-W237, M1-E236, M1-G235, M1-D234, M1-D233,M1-D232, M1-D231, M1-E230, M1-N229, M1-Y228, M1-E227, M1-E226, M1-T225,M1-I224, M1-E223, M1-D222, M1-L221, M1-G220, M1-V219, M1-T218, M1-N217,M1-S216, M1-E215, M1-S214, M1-V213, M1-P212, M1-E211, M1-E210, M1-Q209,M1-S208, M1-E207, M1-Q206, M1-E205, M1-Q204, M1-V203, M1-D202, M1-V201,M1-G200, M1-K199, M1-I198, M1-L197, M1-D196, M1-T195, M1-Q194, M1-P193,M1-T192, M1-E191, M1-D190, M1-I189, M1-T188, M1-K187, M1-D186, M1-N185,M1-S184, M1-L183, M1-E182, M1-N181, M1-P180, M1-E179, M1-E178, M1-T177,M1-E176, M1-L175, M1-G174, M1-G173, M1-K172, M1-P171, M1-K170, M1-P169,M1-S168, M1-F167, M1-T166, M1-P165, M1-L164, M1-L163, M1-G162, M1-K161,M1-K160, M1-R159, M1-L158, M1-E157, M1-A156, M1-K155, M1-E154, M1-R153,M1-Q152, M1-R151, M1-R150, M1-G149, M1-G148, M1-R147, M1-R146, M1-K145,M1-T144, M1-A143, M1-V142, M1-V141, M1-F140, M1-G139, M1-D138, M1-D137,M1-D136, M1-G135, M1-I134, M1-I133, M1-N132, M1-S131, M1-M130, M1-K129,M1-N128, M1-S127, M1-G126, M1-N125, M1-E124, M1-N123, M1-E122, M1-A121,M1-Q120, M1-Q119, M1-N118, M1-K117, M1-L116, M1-V115, M1-E114, M1-G113,M1-P112, M1-F111, M1-S110, M1-R109, M1-L108, M1-N107, M1-D106, M1-E105,M1-G104, M1-N103, M1-N102, M1-L101, M1-C100, M1-E99, M1-L98, M1-E97,M1-Y96, M1-A95, M1-L94, M1-A93, M1-V92, M1-I91, M1-H90, M1-L89, M1-D88,M1-N87, M1-V86, M1-S85, M1-L84, M1-V83, M1-S82, M1-Y81, M1-D80, M1-G79,M1-T78, M1-L77, M1-K76, M1-A75, M1-F74, M1-K73, M1-V72, M1-V71, M1-R70,M1-D69, M1-I68, M1-Y67, M1-E66, M1-Q65, M1-K64, M1-P63, M1-Q62, M1-K61,M1-I60, M1-K59, M1-L58, M1-S57, M1-D56, M1-G55, M1-W54, M1-I53, M1-A52,M1-L51, M1-Q50, M1-Q49, M1-R48, M1-A47, M1-Y46, M1-E45, M1-D44, M1-K43,M1-L42, M1-E41, M1-S40, M1-H39, M1-V38, M1-G37, M1-P36, M1-T35, M1-T34,M1-Y33, M1-Y32, M1-A31, M1-T30, M1-A29, M1-Y28, M1-Q27, M1-Q26, M1-L25,M1-T24, M1-T23, M1-A22, M1-P21, M1-Q20, M1-T19, M1-I18, M1-L17, M1-P16,M1-G15, M1-A14, M1-D13, M1-S12, M1-I11, M1-L10, M1-S9, M1-E8, and/orM1-I7 of SEQ ID NO:21. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal CaYOR056c deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polynucleotide sequence provided in SEQ ID NO:10, andin particular to the coding region of the polynucleotide sequenceprovided in SEQ ID NO:10. Preferably such polynucleotides encodepolypeptides that have biological activity.

The present invention also encompasses polypeptides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polypeptide sequence provided in SEQ ID NO:21.

Most preferred are polypeptides that share at least about 99.5% identitywith the polypeptide sequence provided in SEQ ID NO:21.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:10 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 1384 ofSEQ ID NO:10, b is an integer between 15 to 1398, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ IDNO:10, and where b is greater than or equal to a+14.

Features of the Polypeptide Encoded by Polynucleotide No:11

The polynucleotide sequence (SEQ ID NO:11) and deduced amino acidsequence (SEQ ID NO:22) of the novel fungal essential gene, CaYLR009w(also referred to as FCG17), of the present invention. The CaYLR009wpolypeptide (SEQ ID NO:22) is encoded by nucleotides 1 to 585 of SEQ IDNO:11 and has a predicted molecular weight of 23.1 kDa.

In preferred embodiments, the present invention encompasses apolynucleotide lacking the initiating start codon, in addition to, theresulting encoded polypeptide of CaYLR009w. Specifically, the presentinvention encompasses the polynucleotide corresponding to nucleotides 4thru 585 of SEQ ID NO:11, and the polypeptide corresponding to aminoacids 2 thru 195 of SEQ ID NO:22. Also encompassed are recombinantvectors comprising said encoding sequence, and host cells comprisingsaid vector.

The invention also encompasses N- and/or C-terminal deletions of theCaYLR009w polypeptide of the present invention.

In preferred embodiments, the following N-terminal CaYLR009w deletionpolypeptides are encompassed by the present invention: M1-C195, R2-C195,I3-C195, Y4-C195, Q5-C195, C6-C195, H7-C195, F8-C195, C9-C195, S10-C195,S11-C195, P12-C195, V13-C195, Y14-C195, P15-C195, L16-C195, H17-C195,G18-C195, I19-C195, T20-C195, F21-C195, V22-C195, R23-C195, N24-C195,D25-C195, A26-C195, K27-C195, E28-C195, F29-C195, R30-C195, F31-C195,C32-C195, R33-C195, S34-C195, K35-C195, C36-C195, H37-C195, K38-C195,A39-C195, F40-C195, K41-C195, Q42-C195, R43-C195, R44-C195, N45-C195,P46-C195, R47-C195, K48-C195, L49-C195, R50-C195, W51-C195, T52-C195,K53-C195, A54-C195, F55-C195, R56-C195, K57-C195, A58-C195, A59-C195,G60-C195, K61-C195, E62-C195, L63-C195, V64-C195, V65-C195, D66-C195,S67-C195, T68-C195, L69-C195, T70-C195, F71-C195, A72-C195, A73-C195,R74-C195, R75-C195, N76-C195, V77-C195, P78-C195, V79-C195, R80-C195,Y81-C195, N82-C195, R83-C195, D84-C195, L85-C195, V86-C195, A87-C195,T88-C195, T89-C195, L90-C195, K91-C195, G92-C195, M93-C195, S94-C195,R95-C195, I96-C195, E97-C195, E98-C195, I99-C195, R100-C195, Q101-C195,R102-C195, R103-C195, E104-C195, R105-C195, A106-C195, F107-C195,Y108-C195, K109-C195, N110-C195, R111-C195, M112-C195, K113-C195,G114-C195, N115-C195, K116-C195, E117-C195, R118-C195, Q119-C195,L120-C195, A121-C195, A122-C195, D123-C195, R124-C195, K125-C195,L126-C195, V127-C195, A128-C195, D129-C195, N130-C195, P131-C195,E132-C195, L133-C195, L134-C195, R135-C195, L136-C195, R137-C195,E138-C195, V139-C195, E140-C195, L141C195, R142-C195, R143-C195,K144-C195, A145-C195, E146-C195, K147-C195, L148-C195, A149-C195,A150-C195, K151-C195, E152-C195, N153-C195, A154-C195, M155-C195,E156-C195, E157-C195, D158-C195, E159-C195, E160-C195, T161-C195,E162-C195, V163-C195, E164-C195, E165-C195, E166-C195, G167-C195,E168-C195, G169-C195, D170-C195, E171-C195, E172-C195, M173-C195,I174-C195, S175-C195, G176-C195, E177-C195, E178-C195, E179-C195,W180-C195, E181-C195, S182-C195, E183-C195, D184-C195, E185-C195,S186-C195, E187-C195, R188-C195, and/or E189-C195 of SEQ ID NO:22.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalCaYLR009w deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

In preferred embodiments, the following C-terminal CaYLR009w deletionpolypeptides are encompassed by the present invention: M1-C195, M1-T194,M1-K193, M1-T192, M1-D191, M1-S190, M1-E189, M1-R188, M1-E187, M1-S186,M1-E185, M1-D184, M1-E183, M1-S182, M1-E181, M1-W180, M1-E179, M1-E178,M1-E177, M1-G176, M1-S175, M1-I174, M1-M173, M1-E172, M1-E171, M1-D170,M1-G169, M1-E168, M1-G167, M1-E166, M1-E165, M1-E164, M1-V163, M1-E162,M1-T161, M1-E160, M1-E159, M1-D158, M1-E157, M1-E156, M1-M155, M1-A154,M1-N153, M1-E152, M1-K151, M1-A150, M1-A149, M1-L148, M1-K147, M1-E146,M1-A145, M1-K144, M1-R143, M1-R142, M1-L141, M1-E140, M1-V139, M1-E138,M1-R137, M1-L136, M1-R135, M1-L134, M1-L133, M1-E132, M1-P131, M1-N130,M1-D129, M1-A128, M1-V127, M1-L126, M1-K125, M1-R124, M1-D123, M1-A122,M1-A121, M1-L120, M1-Q119, M1-R118, M1-E117, M1-K116, M1-N115, M1-G114,M1-K113, M1-M112, M1-R111, M1-N110, M1-K109, M1-Y108, M1-F107, M1-A106,M1-R105, M1-E104, M1-R103, M1-R102, M1-Q101, M1-R100, M1-I99, M1-E98,M1-E97, M1-I96, M1-R95, M1-S94, M1-M93, M1-G92, M1-K91, M1-L90, M1-T89,M1-T88, M1-A87, M1-V86, M1-L85, M1-D84, M1-R83, M1-N82, M1-Y81, M1-R80,M1-V79, M1-P78, M1-V77, M1-N76, M1-R75, M1-R74, M1-A73, M1-A72, M1-F71,M1-T70, M1-L69, M1-T68, M1-S67, M1-D66, M1-V65, M1-V64, M1-L63, M1-E62,M1-K61, M1-G60, M1-A59, M1-A58, M1-K57, M1-R56, M1-F55, M1-A54, M1-K53,M1-T52, M1-W51, M1-R50, M1-L49, M1-K48, M1-R47, M1-P46, M1-N45, M1-R44,M1-R43, M1-Q42, M1-K41, M1-F40, M1-A39, M1-K38, M1-H37, M1-C36, M1-K35,M1-S34, M1-R33, M1-C32, M1-F31, M1-R30, M1-F29, M1-E28, M1-K27, M1-A26,M1-D25, M1-N24, M1-R23, M1-V22, M1-F21, M1-T20, M1-I19, M1-G18, M1-H17,M1-L16, M1-P15, M1-Y14, M1-V13, M1-P12, M1-S11, M1-S10, M1-C9, M1-F8,and/or M1-H7 of SEQ ID NO:22. Polynucleotide sequences encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these C-terminal CaYLR009w deletion polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

The present invention also encompasses polynucleotides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polynucleotide sequence provided in SEQ ID NO:11, andin particular to the coding region of the polynucleotide sequenceprovided in SEQ ID NO:11. Preferably such polynucleotides encodepolypeptides that have biological activity.

Most preferred are polynucleotides that share at least about 91.8%identity with the polynucleotide sequence provided in SEQ ID NO:22.

The present invention also encompasses polypeptides sharing at leastleast about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to a the polypeptide sequence provided in SEQ ID NO:22.

Most preferred are polypeptides that share at least about 88.6% identitywith the polypeptide sequence provided in SEQ ID NO:22.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:11 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 571 ofSEQ ID NO:11, b is an integer between 15 to 585, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:11, and where b is greater than or equal to a+14.

Homology Models

One embodiment of the homology models of the present invention utilizesSystem 10 as disclosed in WO 98/11134, the disclosure of which isincorporated herein by reference in its entirety. Briefly, one versionof these embodiments comprises a computer comprising a centralprocessing unit (“CPU”), a working memory which may be, e.g, RAM(random-access memory) or “core” memory, mass storage memory (such asone or more disk drives or CD-ROM drives), one or more cathode-ray tube(“CRT”) display terminals, one or more keyboards, one or more inputlines, and one or more output lines, all of which are interconnected bya conventional bidirectional system bus.

Input hardware, coupled to the computer by input lines, may beimplemented in a variety of ways. Machine-readable data of thisinvention may be inputted via the use of a modem or modems connected bya telephone line or dedicated data line. Alternatively or additionally,the input hardware may comprise CD-ROM drives or disk drives. Inconjunction with a display terminal, keyboard may also be used as aninput device.

Output hardware, coupled to the computer by output lines, may similarlybe implemented by conventional devices. By way of example, outputhardware may include a CRT display terminal for displaying a graphicalrepresentation of a region or domain of the present invention using aprogram such as QUANTA as described herein. Output hardware might alsoinclude a printer, so that hard copy output may be produced, or a diskdrive, to store system output for later use.

In operation, the CPU coordinates the use of the various input andoutput devices, coordinates data accesses from mass storage, andaccesses to and from the working memory, and determines the sequence ofdata processing steps. A number of programs may be used to process themachine-readable data of this invention. Such programs are discussed inreference to the computational methods of drug discovery as describedherein. Specific references to components of the hardware system areincluded as appropriate throughout the following description of the datastorage medium.

For the purpose of the present invention, any magnetic data storagemedium which can be encoded with machine-readable data would besufficient for carrying out the storage requirements of the system. Themedium could be a conventional floppy diskette or hard disk, having asuitable substrate, which may be conventional, and a suitable coating,which may be conventional, on one or both sides, containing magneticdomains whose polarity or orientation could be altered magnetically, forexample. The medium may also have an opening for receiving the spindleof a disk drive or other data storage device.

The magnetic domains of the coating of a medium may be polarized ororiented so as to encode in a manner which may be conventional, machinereadable data such as that described herein, for execution by a systemsuch as the system described herein.

Another example of a suitable storage medium which could also be encodedwith such machine-readable data, or set of instructions, which could becarried out by a system such as the system described herein, could be anoptically-readable data storage medium. The medium could be aconventional compact disk read only memory (CD-ROM) or a rewritablemedium such as a magneto-optical disk which is optically readable andmagneto-optically writable. The medium preferably has a suitablesubstrate, which may be conventional, and a suitable coating, which maybe conventional, usually of one side of substrate.

In the case of a CD-ROM, as is well known, the coating is reflective andis impressed with a plurality of pits to encode the machine-readabledata. The arrangement of pits is read by reflecting laser light off thesurface of the coating. A protective coating, which preferably issubstantially transparent, is provided on top of the reflective coating.

In the case of a magneto-optical disk, as is well known, the coating hasno pits, but has a plurality of magnetic domains whose polarity ororientation can be changed magnetically when heated above a certaintemperature, as by a laser. The orientation of the domains can be readby measuring the polarization of laser light reflected from the coating.The arrangement of the domains encodes the data as described above.

Recombinants and Expression

The present invention provides recombinant DNA molecules containingpolynucleotide sequences encoding essential polynucleotide polypeptides.“Recombinant DNA molecules” include both cloning and expression vectors.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding essential polynucleotide. For example, in one embodiment,routine cloning, subcloning, and propagation of polynucleotide sequencesencoding essential polynucleotides can be achieved using amultifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JollaCalif.) or PSPORT plasmid (Life Technologies). Ligation of sequencesencoding essential polynucleotide into the vector's multiple cloningsite disrupts the lacZ gene, allowing a colorimetric screening procedurefor identification of transformed bacteria containing recombinantmolecules. In addition, these vectors may be useful for in vitrotranscription, dideoxy sequencing, single strand rescue with helperphage, and, creation of nested deletions in the cloned sequence. (See,e.g., Van Heeke, G. and S. M. Schuster J. Biol 264: 5503-5509 (1989)).

In a preferred embodiment, the present invention provides expressionvectors containing a polynucleotide that encodes essentialpolynucleotide polypeptides. Preferably, the expression vectors of thepresent invention comprise polynucleotides that encode polypeptidesincluding the amino acid residue sequences of SEQ ID NO: 12 through SEQID NO: 24.

An “expression vector” refers to an assembly which is capable ofdirecting the expression of desired proteins. The vector must includeregulatory sequences which are operably linked to a gene(s) of interest.The vector may be composed of either DNA or RNA or a combination of thetwo. Optionally, the vector may include a polyadenylation sequence, oneor more restriction sites, as well as one or more selectable markerssuch as neomycin phosphotransferase or hygromycin phosphotransferase.Additionally, depending on the host cell chosen and the vector employed,other genetic elements such as an origin of replication, additionalnucleic acid restriction sites, enhancers, sequences conferringinducibility of transcription and selectable markers, may also beincorporated in the vectors described herein.

The invention provides expression vectors including a polynucleotidedisclosed herein operatively linked to a regulatory sequence.“Regulatory sequences” include enhancers and promoters. Preferably, theexpression vectors of the invention comprise polynucleotide operativelylinked to a prokaryotic promoter. More preferably, the expressionvectors of the present invention comprise a polynucleotide operativelylinked to a eukaryotic promoter, and the expression vectors furthercomprise a polyadenylation signal that is positioned 3′ of thecarboxy-terminal amino acid and within a transcriptional unit of theencoded polypeptide.

A promoter is a region of a DNA molecule typically within about 100nucleotide pairs in front of (upstream of) the point at whichtranscription begins (i.e., a transcription start site). That regiontypically contains several types of DNA sequence elements that arelocated in similar relative positions in different polynucleotides. Asused herein, the term “promoter” includes what is referred to in the artas an upstream promoter region, a promoter region or a promoter of ageneralized eukaryotic RNA Polymerase II transcription unit.

Another type of regulatory sequence is an enhancer. An enhancer providesspecificity of time, location and expression level for a particularencoding region (e.g., gene). A major function of an enhancer is toincrease the level of transcription of a coding sequence in a cell thatcontains one or more transcription factors that bind to that enhancer.Unlike a promoter, an enhancer can function when located at variabledistances from transcription start sites so long as a promoter ispresent.

In one aspect of the invention, the enhancer and/or promoter isoperatively linked to a coding sequence that encodes at least onepolynucleotide product. As used herein, the phrase “operatively linked”means that a regulatory sequence is connected to a coding sequence insuch a way that the transcription of that coding sequence is controlledand regulated by that enhancer and/or promoter. Means for operativelylinking an enhancer and/or promoter to a coding sequence are well-knownin the art. As is also well-known in the art, the precise orientationand location relative to a coding sequence whose transcription iscontrolled, is dependent upon the specific nature of the regulatorysequence. Thus, a TATA box minimal promoter is typically located fromabout 25 to about 30 base pairs upstream of a transcription initiationsite and an upstream promoter element is typically located from about100 to about 200 base pairs upstream of a transcription initiation site.In contrast, an enhancer can be located downstream from the initiationsite and can be at a considerable distance from that site.

Microbial promoters most commonly used in recombinant DNA constructioninclude the beta-lactamase (penicillinase) and lactose promoter systemsand a tryptophan (TRP) promoter system (EPO Appl. Publ. No. 0036776).While these are the most commonly used, other microbial promoters havebeen discovered and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to introducefunctional promoters into plasmid vectors.

Suitable promoter sequences in yeast vectors include the promoters for3-phosphoglycerate kinase or other glycolytic enzymes such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesepolynucleotides are also introduced into the expression vectordownstream from the sequences to be expressed to provide polyadenylationof the mRNA and termination. Other promoters, which have the additionaladvantage of transcription controlled by growth conditions, are thepromoter region for alcohol dehydrogenase 2, isocytochrome C, acidphosphatase, degradative enzymes associated with nitrogen metabolism,and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, andenzymes responsible for maltose and galactose utilization. Any plasmidvector containing a yeast-compatible promoter, origin or replication andtermination sequences is suitable.

In one embodiment, a regulatable promoter is used. By “regulatablepromoter” is meant a promoter other than the native promoter for theessential polynucleotide which can be regulated by the addition and/orremoval of specific materials or for example by other environmentalchanges.

Some examples of regulatable promoters for use in organisms include GALI for use in S. cerevisiae (repressed by glucose induced by galactose);NMT1 for use in S. pombe (repressed by thiamine); for use in C.albicans: MALI (repressed by glucose, induced by maltose, sucrose); foruse in E. coli: araB (repressed by glucose, induced by arabinose); foruse in Gram-positive bacteria such as Staphylococci, Enterococci,Streptococci and Bacilli: xylA/xylR (from S. xylosus) (repressed byglucose, induced by xylose); for use in E. coli and B. subtilis pSPAC(an artificial promoter derived from E. coli lac, regulated by IPTG, seeVagner et al. Microbiology, 144, 3097-3104 (1998)); and for all of theabove organisms plus further unspecified fungi, bacteria and mammaliancell lines: tetA/tetR (from various bacterial tetracycline resistancecassettes) this system exists in various versions, see Gossen et al.Current Opin. Biotechnol. 5, pp 516-520 (1994), that are repressible orinducible by various tetracycline analogues.

Other conditional regulatable promoters include, but are not limited to,those such as MET25, MAL2, PHO5,5 GAL I; STE2, or STE3.

A preferred regulatable promoter for use in some embodiments includesthe MET3 promoter (repressible by methionine, cysteine or both).

For use in mammalian cells, the control functions on the expressionvectors are often derived from viral material. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, Cytomegalovirusand most frequently Simian Virus 40 (SV40). The early and late promotersof SV40 virus are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication. Smaller or larger SV40 fragments can also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BglI site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired polynucleotide sequence, provided such control sequences arecompatible with the host cell systems.

An origin of replication can be provided constructing the vector toinclude an exogenous origin, such as can be derived from SV40 or otherviral (e.g., Polyoma, Adeno, VSV, BPV, CMV) source, or can be providedby the host cell chromosomal replication mechanism. If the vector isintegrated into the host cell chromosome, the latter is oftensufficient.

A coding sequence of an expression vector is operatively linked to atranscription terminating region. RNA polymerase transcribes an encodingDNA sequence through a site where polyadenylation occurs. Typically, DNAsequences located a few hundred base pairs downstream of thepolyadenylation site serve to terminate transcription. Those DNAsequences are referred to herein as transcription-termination regions.Those regions are required for efficient polyadenylation of transcribedmessenger RNA (mRNA). Transcription-terminating regions are well-knownin the art.

The invention provides an expression vector having a polynucleotide thatencodes an essential polynucleotide polypeptide. Such a polypeptide ismeant to include a sequence of nucleotide bases encoding an essentialpolynucleotide polypeptide sufficient in length to distinguish saidfragment from a polynucleotide fragment encoding a non-essentialpolynucleotide polypeptide. A polypeptide of the invention can alsoencode biologically functional polypeptides or peptides which havevariant amino acid sequences, such as with changes selected based onconsiderations such as the relative hydropathic score of the amino acidsbeing exchanged. These variant sequences are those isolated from naturalsources or induced in the sequences disclosed herein using a mutagenicprocedure such as site-directed mutapolynucleotidesis.

The present invention also provides recombinant vectors that may be usedto integrate exogenously provided sequences into the genome of a hostcell. The recombinant integration vectors of the present inventioninclude a polynucleotide that encodes a selectable marker andpolynucleotide sequences of the invention or fragment thereof. Theintegration vectors are used to integrate the essential polynucleotidesequences into a target polynucleotide sequence that resides within thefungal host genome (e.g., endogenous sequence), thereby disrupting thefunction of the target polynucleotide sequence within the fungal cells.These integration vectors may be used in a polynucleotide disruptionassay to screen candidate sequences in order to identify the candidatesequences that encode a polynucleotide product that is required forfungal cell viability.

Accordingly, these recombinant integration vectors include candidatesequences such as homologues of SEQ ID NO: 1 through to SEQ ID NO: 11,SEQ ID NO: 1 through to SEQ ID NO. 11, SEQ ID NO: 12 or a fragmentthereof to determine if the candidate sequences encode a polynucleotideproduct that is required for cell viability. The candidate sequencesthat is included as part of the recombinant integration vector is the“exogenous” candidate sequence that is employed as the “disrupting”sequence in a polynucleotide disruption assay. The candidate sequencethat resides within the host genome is the “endogenous” or targetcandidate sequence.

The integration event rarely occurs, for example, by non-homologousrecombination in which a recombinant vector, that includes the exogenouscandidate sequence, inserts the exogenous candidate sequence into arandom location within the host genome.

In a more preferred embodiment, the integration event inserts theexogenous candidate sequence into a specific target site within the hostgenome. The targeted integration event can involve homologousrecombination in which the integration vector, that includes theexogenous candidate sequence, inserts the exogenous candidate sequenceinto its homologous target candidate sequence that resides within thehost's genome (e.g., the endogenous candidate sequence). The exogenouscandidate sequences can result in disruption of the function of theendogenous candidate sequence. For example, disrupting the function ofthe endogenous sequence may result in the loss of fungal cell viability.

Host Cells and Host Organisms

In yet another embodiment, the present invention provides recombinanthost cells transformed or transfected with a polynucleotide that encodesessential polynucleotide polypeptides, as well as transgenic cellsderived from those transformed or transfected cells. Preferably, therecombinant host cells of the present invention are transfected with thepolynucleotide of SEQ ID NO: 1 to SEQ ID NO: 11, or a variant orfragment thereof.

A variety of cells are amenable to the method of the invention involvingpolypeptide expression, for instance, yeast cells, human cell lines, andother eukaryotic cell lines well-known to those of skill in the art.

Means of transforming or transfecting cells with an exogenouspolynucleotide such as DNA molecules are well-known in the art andinclude techniques such as calcium-phosphate- or DEAE-dextran-mediatedtransfection, protoplast fusion, electroporation, liposome mediatedtransfection, direct microinjection and adenovirus infection (Sambrook,et al., supra).

The most widely used method is transfection mediated by either calciumphosphate or DEAE-dextran. Although the mechanism remains obscure, it isbelieved that the transfected DNA enters the cytoplasm of the cell byendocytosis and is transported to the nucleus. Depending on the celltype, up to 90% of a population of cultured cells can be transfected atany one time. Because of its high efficiency, transfection mediated bycalcium phosphate or DEAE-dextran is the method of choice forexperiments that require transient expression of foreign DNA in largenumbers of cells. Calcium phosphate-mediated transfection is also usedto establish cell lines that integrate copies of foreign DNA, which areusually arranged in head-to-tail tandem arrays into the host cellgenome.

In the protoplast fusion method, protoplasts derived from bacteriacarrying high numbers of copies of a plasmid of interest are mixeddirectly with cultured mammalian cells. After fusion of the cellmembranes (usually with polyethylene glycol), the contents of thebacteria are delivered into the cytoplasm of the mammalian cells and theplasmid DNA is transported to the nucleus. Protoplast fusion frequentlyyields multiple copies of the plasmid DNA tandemly integrated into thehost chromosome.

The application of brief, high-voltage electric pulses to a variety ofmammalian cells leads to the formation of nanometer-sized pores in theplasma membrane. DNA is taken directly into the cell cytoplasm eitherthrough these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of cloned polynucleotides and for establishment ofcell lines that carry integrated copies of the polynucleotide ofinterest. Electroporation, in contrast to calcium phosphate-mediatedtransfection and protoplast fusion, frequently gives rise to cell linesthat carry one, or at most a few, integrated copies of the foreign DNA.

Liposome transfection involves encapsulation of DNA and RNA withinliposomes, followed by fusion of the liposomes with the cell membrane.The mechanism of how DNA is delivered into the cell is unclear buttransfection efficiencies can be as high as 90%.

Direct microinjection of a DNA molecule into nuclei has the advantage ofnot exposing DNA to cellular compartments such as low-pH endosomes.Microinjection is therefore used primarily as a method to establishlines of cells that carry integrated copies of the DNA of interest.

The use of adenovirus as a vector for cell transfection is well-known inthe art. Adenovirus vector-mediated cell transfection has been reportedfor various cells (Stratford-Perricaudet, et al., J Clin Invest.90(2):626-630).

A transfected cell can be prokaryotic or eukaryotic. In one embodiment,the host cells of the invention are eukaryotic host cells.

When the recombinant host cells of the present invention are prokaryotichost cells Escherichia coli bacterial cells are preferred. In general,prokaryotes are preferred for the initial cloning of DNA sequences andconstructing the vectors useful in the invention. For example, E. coliK12 strains can be particularly useful. Another microbial strain whichcan be used includes E. coli X1776 (ATCC No. 31537). These examples are,of course, intended to be illustrative rather than limiting.

Prokaryotes can also be used for expression. The aforementioned strains,as well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325),bacilli such as Bacillus subtilis, or other enterobacteriaceae such asSalmonella typhimurium or Serratus marcesens, and various Pseudomonasspecies can be used.

In addition to prokaryotes, eukaryotic microbes such as yeast can alsobe used. Saccharomyces cerevisiae or common baker's yeast is the mostcommonly used among eukaryotic microorganisms, although a number ofother strains are commonly available. For expression in Saccharomyces,the plasmid YRp7, for example, is used. This plasmid already containsthe trpl polynucleotide which provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for example,ATCC No. 44076. The presence of the trpl lesion as a characteristic ofthe yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

In addition to microorganisms, cultures of cells derived frommulticellular organisms can also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. Examples of such useful host cell lines are AtT-20, VERO andHeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK,COSM6, COS-7, 293 and MDCK cell lines.

Following transfection, the cell is maintained under culture conditionsfor a period of time sufficient for expression of an essentialpolynucleotide polypeptide. Culture conditions are well-known in the artand include ionic composition and concentration, temperature, pH and thelike. Typically, transfected cells are maintained under cultureconditions in a culture medium. Suitable medium for various cell typesare well-known in the art. In a preferred embodiment, temperature isfrom about 20° C. to about 50° C., more preferably from about 30° C. toabout 40° C. and, even more preferably, about 37° C. pH is preferablyfrom about a value of 6.0 to a value of about 8.0, more preferably fromabout a value of about 6.8 to a value of about 7.8 and, most preferably,about 7.4. Osmolality is preferably from about 200 milliosmols per liter(mosm/L) to about 400 mosm/L and, more preferably, from about 290 mosm/Lto about 310 mosm/L. Other biological conditions needed for transfectionand expression of an encoded protein are well-known in the art.

Transfected cells are maintained for a period of time sufficient forexpression of an essential polynucleotide polypeptide. A suitable timedepends upon the cell type used and is readily determinable by a skilledartisan. Typically, maintenance time is from about 2 to about 14 days.Recombinant essential polynucleotide polypeptide is recovered orcollected either from the transfected cells or the medium in which thosecells are cultured. Recovery comprises isolating and purifying theessential polynucleotide polypeptide. Isolation and purificationtechniques for polypeptides are well-known in the art and include suchprocedures as precipitation, filtration, chromatography, electrophoresisand the like.

Fungal Strains of the Invention

The invention also provides Candida albicans strains which may be usedfor drug screening. According to the invention, one copy of an essentialpolynucleotide of the invention is eliminated (such as those encoding anessential polypeptide having the amino acid sequence of SEQ ID NO:12through to SEQ ID NO 22), while the second allele is placed under thecontrol of a regulatable promoter.

In a preferred embodiment, precise replacement of one copy of a targetpolynucleotide is facilitated by using a PCR-based polynucleotidedisruption tool (see, e.g., Wilson et al., J. Bacteriol. 181:1868-1874(1999), herein incorporated by reference). Genes are disrupted by usingPCR to generate a selectable marker (for example, URA3) surrounded bythe 5′ and 3′ sequences of the polynucleotide to be disrupted. Themarker sequences are part of a plasmid (see, e.g., Example 4).Disruption cassettes are synthesized by PCR using primers containingboth short flanking homology (SFH) regions and regions which anneal tothe marker in the plasmid. The SHF regions are about 25 to about 60 bplong. The disruption cassettes are generated in one-step PCR synthesisand homologous recombination transplaces the recombinant null constructinto the genome, generating an allele such as yfg1::URA3 (yfg1 is “yourfavorite gene”).

Where the polynucleotide is essential, elimination of both alleles willbe lethal or severely crippling for growth. Therefore, in the presentinvention, a regulatable promoter is used to provide a range of levelsof expression of the second allele. Depending on the conditions, thesecond allele can be non-expressing, underexpressing, or expressing at anormal level relative to that when the allele is linked to its nativepromoter.

Regulatable promoters include, but are not limited to, those such asMET25, MAL2, PHO5,5 GAL I; STE2, or STE3. A preferred regulatablepromoter is the MET3 promoter.

Preferably, replacement of the promoter of the second copy with the MET3promoter is accomplished by the use of a promoter swapping cassette. The“PCR-based promoter swapping cassette” as used herein refers to acassette comprised of a regulatable promoter, a selectable marker, andshort flanking regions to on the 5′ and 3′ ends of the cassette whichare homologous to the native promoter and the start of thepolynucleotide coding region, respectively.

This promoter swapping cassette works analogously to the PCR basedpolynucleotide disruption cassette described above. A promoter of ‘YFG’is disrupted by using PCR to generate a selectable marker (for example,URA3 or ARG4) containing a sequence of the promoter region to bedisrupted. Disruption cassettes are synthesized by PCR, using shortflanking homology (SFH) regions to the promoter of interest. SHF regionsare about 25 to about 60 bp long. When the disruption cassette istransformed into yeast cells, the promoter of YFG is displaced byhomologous recombination.

In order to replace the endogenous promoter of YFG, the PCR-basedpromoter swapping cassette contains a selective marker amplified from aplasmid using a pair of primers designed so that the forward primercontains about 50 to about 60 bp of flanking sequences that are derivedfrom sequences 500-100 bp upstream of the ATG codon of YFG to ensure itwill anneal upstream or on the boundary of the endogenous promoter andthis portion of the forward primer is attached to the 5′-end of theforward common promoter primer. The forward common promoter primerregion is common to the plasmid.

The reverse primer has 20 to 60 or preferably about 50 to about 60 bp offlanking sequences, which are derived from the start codon region of thepolynucleotide or ORF including ATG attached to the 3′ end of thereverse common primer. The resulting PCR product contains the promotercassette that is flanked by about 50 to about 60 bp of sequences, oneither end, homologous to the upstream promoter region and to the codingregion of the polynucleotide of interest, respectively. Once introducedinto the cells heterozygous for the polynucleotide of interest obtainedvia the regular PCR-based polynucleotide disruption approach, thePCR-based promoter swapping cassette would replace the endogenouspromoter of the remaining allele via homologous recombination.

The MET3 promoter for use in the construction of the C. albicans strainsas described above is a homologue of S. cervisiae MET3. The cloning ofthis promoter is described in Care et al., supra). The MET3polynucleotide of S. cervisiae encodes ATP sulphurylase (ATP: sulphateadenyltransferase, E.C. 2.7.7.4) which catalyses the production ofadenosine 5′-phosphosulphate (APS) from inorganic sulphate and ATP, thefirst step in the assimilation of inorganic sulphate. Expression of MET3is repressed by exogenous methionine and S-adenysl methionine (SAM).Methionine is converted into SAM, which is thought to be the truerepressor. Sulphur assimilation is also required for the biosynthesis ofcysteine. According to an alternative view, cysteine is the truerepressor of the enzymes of sulphur metabolism, the action of methionineand SAM being dependent on the interconversion of sulphur-containingamino acids through transulphurylation pathways (Care et al. supra,herein incorporated by reference).

In the methods of the invention, the MET3 promoter may be completely orpartially repressed using cysteine, methionine or both amino acids.

In particular, the present invention encompasses strains of Candidaalbicans cells in which both alleles of a polynucleotide are modified. Afirst copy of a polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of one of SEQ ID NO 1 to 11 is inactive and asecond copy of the polynucleotide is under the control of a regulatablepromoter.

Additionally, the invention also provides a strain of Candida albicanshaving a nucleic acid molecule comprising a nucleotide sequence selectedfrom one of SEQ ID NO: 1 to 11 under the control of a regulatablepromoter.

Target Evaluation in Animal Model Systems

In one embodiment, the essential strains provided by the invention areused in animal studies to examine the effect of polynucleotideinactivation by conditional expression. Animal studies, using mice, forexample, may be inoculated with one or more of the strains of theinvention. In a more desirable embodiment of the invention, the effecton mice injected with a lethal inoculum of one of the essential strainscould be determined depending on whether the mice were provided with anappropriate concentration of promoter repressor to inactivate expressionof a drug target polynucleotide. The lack of expression of apolynucleotide demonstrated to be essential under laboratory conditionscan thus be correlated with prevention of a terminal C. albicansinfection. In this type of experiment, only mice “treated” with promoterrepressor supplements are predicted to survive infection becauseinactivation of the target polynucleotide has killed the essentialstrain pathogen within the host.

Identification of Essential Genes

Also provided herein are methods to identify essential polynucleotides.In order to determine essentially, a strain is constructed as describedabove and then the strain is cultured under conditions wherein thesecond modified allele of the polynucleotide which is under conditionalexpression is substantially underexpressed or not expressed. A “promoterrepressor”, i.e., a substance that inhibits the ability of a regulatablepromoter to cause expression in the second allele is added to theculture medium. The preferred promoter repressor used herein may bemethionine and cysteine or a combination thereof when the MET3 promoteris used in the compositions and methods of the invention. The preferredregulatable promoter is MET3. The viability and/or growth of the strainis compared with that of control cells cultured without the addition ofpromoter supressor. A loss or reduction of viability or growth in thecells cultured with promoter suppressor indicates that thepolynucleotide is essential to the survival of the fungus.

The fungal strains and cells used to identify essential polynucleotideswith the method of the invention, include but are not limited to Absidiacorymbigera, Aspergillusflavis, Aspergillus fumigatus, Aspergillusniger, Botrytis cinerea, Candida dublinensis, Candida glabrata, Candidakrusei, Candia parapsilopsis, Candia tropicalis, Coccidioides immitis,Cryptococcus neoformians, Erysiphe graminis, Exophalia dernatiditis,Fusarium osysproum, Histoplasma capsulatum, Magnaporthe grisea, Mucorrouxii, Pneumocystis carinii, Puccinia graminis, Puccinia recodita,Rhizomucor pusillus, Puccinia striiformis, Rhizopus arrhizus, Septoriaavenae, Septoria nodorum, Septoria triticii, Tilletia controversa,Tilletia tritici, Trichospoon beigelii and Ustilago maydis. Preferably,Candida albicans strains are used.

Haploid or diploid strains may used to identify essentialpolynucleotides by the method of the invention. In the case of haploidstrains, the promoter of the polynucleotide of interest is replaced witha regulatable promoter and tested for essentiality as described herein.Since there is no diploidy, the first step using a PCR based disruptioncassette is not required.

The nucleotide sequences encoding candidate polynucleotides which areassayed using the method of the invention to determine essentiality arepreferably conserved polynucleotides. A polynucleotide can be identifiedas belonging to a repertoire of conserved polynucleotides using severalmethods. For example, an isolated polynucleotide may be used as ahybridization probe under low stringency conditions to detect othermembers of the repertoire of conserved polynucleotides present ingenomic DNA using the methods described by Southern, J. Mol. Biol.,98:503 (1975). Additionally, conserved polynucleotides can be identifiedusing a concordance analysis such as that described herein.

Strains Hypersensitive to Drugs and Titration of Gene Products

Also, provided herein are methods to create strains which arehypersensitive to potential antifungal drugs. Current cell based assaysused to identify or to characterize compounds for drug discovery anddevelopment frequently depend on detecting the ability of a testcompound to modulate the activity of a target molecule located within acell or located on the surface of a cell. Most often such targetmolecules are proteins such as enzymes, receptors and the like. However,target molecules also include other molecules such as DNAs, lipids,carbohydrates and RNAs including messenger RNAs, ribosomal RNAs, tRNAsand the like. A number of highly sensitive cell-based assay methods areavailable to those of skill in the art to detect binding and interactionof test compounds with specific target molecules. However, these methodsare generally not highly effective when the test compounds binds to orotherwise interacts with its target molecule with moderate or lowaffinity. Thus, current cell-based assay methods are limited in thatthey are not effective in identifying or characterizing compounds thatinteract with their targets with moderate to low affinity or compoundsthat interact with targets that are not readily accessible.

The methods of the invention to create cells which are hypersensitive topotential antifungal compounds may be used to overcome theselimitations. The sensitizing assays of the present invention are capableof detecting compounds exhibiting low or moderate potency against thetarget molecule of interest because such compounds are substantiallymore potent on sensitized cells than on non-sensitized cells. The effectmay be such that a test compound may be two to several times morepotent, at least 10 times more potent, at least 20 times more potent, atleast 50 times more potent, at least 100 times more potent, at least1000 times more potent, or even more than 1000 times more potent whentested on the sensitized cells as compared to the non-sensitized cells.

Such assays are useful to identify compounds that previously would nothave been readily identified. A target which expresses a significantamount of product may not result in a particular effect by a particularcompound. However, when the amount of product is reduced, the compoundmay be revealed to in fact have an effect on the polynucleotide product.An initial hit compound which exhibits moderate or even low potency maybe able to be developed into a drug. For example, once a hit compound isidentified with low or moderate potency, a combinatorial chemicallibrary consisting of compounds with structures related to the hitcompound but containing systematic variations including additions,subtractions and substitutions of various structural features may beincluded. When tested for activity against the target molecule,structural features may be identified that either alone or incombination with other features enhance or reduce activity. Thisinformation may be used to design subsequent directed librariescontaining compounds with enhanced activity against the target molecule.After one or several iterations of this process, compounds withsubstantially increased activity against target molecules are identifiedand may be further developed as drugs. This process is facilitated bythe use of the sensitized strains of the present invention sincecompounds acting at the selected targets exhibit increased potency insuch cell-based assays, thus, more compounds can now be characterizedproviding more useful information than would be obtained otherwise.

The method of sensitizing a cell entails selecting an essentialpolynucleotide such as those identified in the present invention. Thenext step is to obtain a cell in which the level or activity of thetarget can be reduced to a level where it is rate limiting forviability. For example, the cell may be a strain of the presentinvention in which the selected polynucleotide is under the control of aMET3 promoter. The amount of RNA transcribed from the selectedpolynucleotide is limited by varying the concentration of methionine,cysteine or both, which acts on the MET3 promoter, thereby varying theactivity of the promoter driving transcription of the RNA. Thus, cellsare sensitized by exposing them to a repressor concentration thatresults in an RNA level such that the function of the selectedpolynucleotide product becomes rate limiting for fungal growth, survivalor proliferation.

In one embodiment of the present invention, a Candida strain is createdby inactivating one copy of a polynucleotide by the insertion of anucleotide sequence encoding a selectable marker and the secondpolynucleotide copy has been modified by recombination with a promoterswapping cassette, to place the second copy under the controlledexpression of a MET3 promoter. The strain is then grown under a firstset of conditions where the MET3 promoter is expressed at a relativelylow level and the extent of growth is determined. This measurement maybe carried out using any appropriate standard known to those skilled inthe art, including optical density, wet weight of pelleted cells, totalcell count, viable count, DNA content and the like. This experiment isrepeated in the presence of a test compound and a second measurement ofgrowth is obtained. The estimate of growth in the presence and in theabsence of the test compound, which can conveniently be expressed interms of indicator values, are then compared. A dissimilarity in theextent of growth or indicator values provides an indication that thetest compound may interact with the target essential polynucleotideproduct.

To gain additional information, additional experiments may be performedin various embodiments. For example, using a second set ofnon-repressing growth conditions where the second polynucleotide copy,under the control of the MET3 promoter, is expressed at various levelshigher that in the rest set of conditions described above. The extent ofgrowth or indicator values is determined in the presence and absence ofthe test compound under this second set of conditions. The extent ofgrowth or indicator values in the presence and in the absence of thetest compound are then compared. A dissimilarity in the extent of growthor indicator values provides an indication that the test compounds mayinteract with the target essential polynucleotide product.

Furthermore, the extent of growth in the first and in the second set ofgrowth conditions can also be compared. If the extent of growth isessentially the same, the data suggest that the test compound does notinhibit the polynucleotide product encoded by the modified allelicpolynucleotide pair carried by the strain tested. However, if the extentof growth is substantially different, the data indicate that the levelof expression of the subject polynucleotide product may determine thedegree of inhibition by the test compound and therefore it is likelythat the subject polynucleotide product is the target of that testcompound.

In one embodiment, the strains of the invention in which the sequencerequired for fungal growth, survival or proliferation of Candidadescribed herein is under the control of MET3, and grown in the presenceof a concentration of promoter repressor which causes the function ofthe polynucleotide products encoded by these sequences to be ratelimiting for fungal growth. To achieve that goal, a growth inhibitiondose curve is calculated by plotting various doses of repressor againstcorresponding growth inhibition caused by the limited levels of thepolynucleotide product required for fungal proliferation. From thisdose-response curve, conditions providing various growth rates for 1 to100%, as compared to repressor-free growth, can be determined. Forexample, the diploid fungal strains of the invention are grown in mediumcomprising a range of methionine concentrations to obtain the growthinhibitory response curve for each strain. First, seed cultures of thediploid fungal strains of the invention are grown in the appropriatemedium. Subsequently, aliquots of the seed cultures are diluted intomedium containing varying concentrations of methionine. For example, thestrains may be grown in duplicate cultures containing two-fold serialdilutions of methionine Additionally, control cells are grown induplicate without methionine. The control cultures are started fromequal amounts of cells derived from the same initial seed culture of thestrain of interest. The cells are grown for an appropriate period oftime and the extent of growth is determined using any appropriatetechnique For example, the extent of growth may be determined bymeasuring the optical density of the cultures. When the control culturereaches mid-log phase the percent growth (relative to the controlculture) for each of the methionine containing cultures is plottedagainst the log concentrations of methionine to produce a growthinhibitory dose response curve for methionine. The concentration ofmethionine that inhibits cell growth at 50% (IC50) as compared to the 0mM methionine control (0% growth inhibition) is then calculated from thecurve. Alternative methods of measuring growth are also contemplated.Examples of these methods include measurements of protein, theexpression of which is engineered of the cells being tested and canreadily be measured.

Thus, in one embodiment, the method described above may be used totitrate the amount of essential polynucleotide product expressed in adiploid fungal cell.

In another embodiment, a homologue of the essential polynucleotidesequences of the present invention that are identified in a haploidorganism may similarly be used as the basis for detection of anantifungal or therapeutic agent. In this embodiment, the test organism(e.g., Aspergillus fumigatus or Cryptococcus neoformans) or any otherhaploid organism in a strain constructed by modifying the single alleleof the target polynucleotide in one step recombination with a promoterswapping cassette such that the expression of the polynucleotide isconditionally regulated by the promoter. Like individual diploid strainsof the invention, sensitized haploid cells may be similarly used inwhole cell-based assay methods to identify compounds displaying apreferential activity against the affected target.

In various embodiments, the modified strain is grown under a first setof conditions where the regulatable promoter is expressed at arelatively low level and the extent of growth determined. Thisexperiment is repeated in the presence of a test compound and a secondmeasurement of growth obtained. The extent of growth in the presence andin the absence of the test compounds are then compared to provide afirst indicator value. Two further experiments are performed usingnon-repressing growth conditions where the target polynucleotide isexpressed at substantially higher levels than in the first set ofconditions. Extent of growth is determined in the presence and absenceof the test compound under the second set of conditions to obtain asecond indicator value. The first and second indicator values are thencompared. If the indicator values are essentially the same, the datasuggest that the test compound does not inhibit the test target.However, if the two indicator values are substantially different, thedata indicate that the level of expression of the target polynucleotideproduct may determine the degree of inhibition by the test compounds andtherefore it is likely that the polynucleotide product is the target ofthat test compound. Whole-cell assays comprising collections or subsetsof multiple sensitized strains may be screened, for example, in a seriesof 96 well, 384 well or even 1586 well microtiter plates.

Cells to be assayed are exposed to the above-determined concentrationsof methionine or other promoter repressor. The presence of the repressorat this sub-lethal concentration reduces the amount of theproliferation-required polynucleotide product to the lowest amount inthe cell that will support growth. Cells grown in the presence of thisconcentration of repressor are more sensitive to inhibitors of theproliferation-required protein or RNA of interest as well as toinhibitors of proteins or RNAs in the same biological pathway as theproliferation-required protein or RNA of interest but not specificallymore sensitive to inhibitors of unrelated proteins or RNAs.

Cells pretreated with sub-inhibitory concentrations of repressors whichtherefore contain a reduced amount of proliferation-required targetpolynucleotide product are used to screen for compounds that reduce cellgrowth. The sub-lethal concentration of repressor may be anyconcentration consistent with the intended use of the assay to identifycandidate compounds to which the cells are more sensitive than arecontrol cells in which this polynucleotide product is not rate-limiting.For example, the sub-lethal concentration of the repressor may be suchthat growth inhibition is at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 75%, at least 80%, at least 90%,at least 95% or more than 96%. Cells which are pre-sensitized using thepreceding method are more sensitive to inhibitors of the target proteinbecause these cells contain less target protein to inhibit thanwild-type cells. Cells are then contacted with a candidate compound andgrowth of the cells in the methionine containing medium is compared togrowth of the control cells in medium which lacks methionine todetermine whether the candidate compound inhibits growth of thesensitized cells (i.e., cells grown in the presence of methionine) to agreater extent than the candidate compound inhibits the growth of cellsgrown in the absence of methionine. For example, if a significantdifference in growth is observed between the sensitized cells and thenon-sensitized cells, the candidate compound may be used to inhibit theproliferation of the organism or may be further optimized to identifycompounds which have an even greater ability to inhibit the growthsurvival or proliferation of the organism.

When screening for antimicrobial agents against a polynucleotide productrequired for fungal growth, survival or proliferation or growthinhibition of cells containing a limiting amount of that polynucleotideproduct can be assayed. Growth inhibition can be measured by directlycomparing the amount of growth measured by the optical density of theculture relative to uninoculated growth medium between and experimentalsample and a control sample. Alternative methods for assaying cellproliferation include measuring green fluorescent protein reportconstruct emissions, various enzymatic activity assays and other methodswell-known in the art.

It will be appreciated that the above cell-based assays may be used toidentify compounds which inhibit the activity of polynucleotide productsfrom organisms other than Candida albicans which are homologous to theCandida albicans nucleotide sequences encoding essential polypeptidesdescribed herein. For example, the nucleotide sequences encodingpolypeptides may be from animal fungal pathogens such as Aspergillusfumigatus, Aspergillus niger, Aspergillus flavis, Candida tropicalis,Candida parapsilopsis, Candida krusei, Cryptococcus neofomras,Coccidioides immitis, Exophalia dermatiditis, Fusarium oxygporum,Histoplasma capsulaturm, Pneumocystis carinii, Trichosporan beigelii,Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus or Absidiacorymbigera or the plant fungal pathogens such as Botrytis cinerea,Erysiphe graminis, Magnaporthe grisea, Puccinia recodita, Spetoriatriticii, Tilletia controversa, Ustilago maydis or any species fallingwith the genera of any of the above species. In some embodiments, theessential polynucleotides are from an organism other than Saccharomycescerevisiae.

Protein Based Assays

The present invention also provides methods for identifying anantimycotic compound comprising screening a plurality of compounds toidentify a compound that modulates the activity or level of apolynucleotide product (mRNA or protein), said polynucleotide productbeing encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 11, or a nucleotide sequencethat is the homologue of a polynucleotide having a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11.

Binding assays may be used to identify antimycotic compounds. Theseassays involve preparing a reaction mixture comprising the targetpolynucleotide product and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex which is removed and/or detected within the reactionmixture. These assays may be conducted in a variety of ways. Forexample, one method involves anchoring a target polynucleotide productor the test substance onto a solid phase and detecting targetpolynucleotide product/test compound complexes anchored, via theintermolecular binding reaction to the solid phase at the end of thereaction. In one embodiment, the target polynucleotide product isanchored onto a solid surface and the test compound which is notanchored is labeled either directly or indirectly.

Microtiter plates may be utilized as the solid phase. The anchoredcomponent is immobilized by non-covalent or covalent attachments.Non-covalent attachment can be accomplished by simply coating the solidsurface with a solution of the protein and drying the coated surface.Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized is used to anchorthe protein to the solid surface.

Alternatively, a reaction is conducted in a liquid phase, the reactionproducts are separated from unreacted components, and complexes aredetected; e.g., using an immobilized antibody specific for the targetpolynucleotide product or for the test compound, to anchor complexesformed in solution, and a second labeled antibody, specific for theother component of the complex to allow detection of anchored complexes.

In another aspect of the invention, methods are employed to fordetecting protein-protein interactions for identifying novel targetprotein-cellular or extracellular protein interactions. Any suitablemethod may be used.

The target polynucleotide products of the invention may interact, invivo, with one or more cellular or extracellular macromolecules, such asproteins. Such macromolecules include, but are not limited to, nucleicacid molecules and proteins identified via methods such as thosedescribed above. For purposes of this discussion, such cellular andextracellular macromolecules are referred to herein as “bindingpartners.” Compounds that disrupt such interactions can be useful inregulating the activity of the target polynucleotide protein, especiallymutant target polynucleotide proteins. Such compounds include, but arenot limited to molecules such as antibodies, peptides, and the like.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the target polynucleotide productand its cellular or extracellular binding partner or partners involvespreparing a reaction mixture containing the target polynucleotideproduct and the binding partner under conditions and for a timesufficient to allow the two to interact and bind, thus forming acomplex. In order to test a compound for inhibitory activity, thereaction mixture is prepared in the presence and absence of the testcompound. The test compound is initially included in the reactionmixture, or added at a time subsequent to the addition of targetpolynucleotide product and its cellular or extracellular bindingpartner. Control reaction mixtures are incubated without the testcompound. The formation of complexes between the target polynucleotideprotein and the cellular or extracellular binding partner is thendetected. The formation of a complex in the control reaction, but not inthe reaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the target polynucleotideprotein and the interactive binding partner. Additionally, complexformation within reaction mixtures containing the test compound andnormal target polynucleotide protein can also be compared to complexformation within reaction mixtures containing the test compound and amutant target polynucleotide protein. This comparison can be importantin those cases wherein it is desirable to identify compounds thatdisrupt intermolecular interactions involving mutant but not normaltarget polynucleotide proteins.

The assay for compounds that interfere with the interaction of thetarget polynucleotide products and binding partners is conducted ineither a heterogeneous or a homogeneous format.

Heterogeneous assays involve anchoring either the target polynucleotideproduct or the binding partner onto a solid phase and detectingcomplexes anchored on the solid phase at the end of the reaction. Inhomogeneous assays, the entire reaction is carried out in a liquidphase. In either approach, the order of addition of reactants is variedto obtain different information about the compounds being tested. Forexample, test compounds that interfere with the interaction between thetarget polynucleotide products and the binding partners, e.g., bycompetition, are identified by conducting the reaction in the presenceof the test substance; i.e., by adding the test substance to thereaction mixture prior to or simultaneously with the targetpolynucleotide protein and an interacting cellular or extracellularbinding partner. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, are tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are described briefly below.

In a heterogeneous assay system, either the target polynucleotideprotein or the interactive cellular or extracellular binding partner, isanchored onto a solid surface, while the non-anchored species islabeled, either directly or indirectly. In practice, microtiter platesare conveniently utilized. The anchored species is immobilized either bynon-covalent or covalent attachment.

Non-covalent attachment is accomplished simply by coating the solidsurface with a solution of the target polynucleotide product or bindingpartner and drying the coated surface. Alternatively, an immobilizedantibody specific for the species to be anchored is used to anchor thespecies to the solid surface. The surfaces can be prepared in advanceand stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface isaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, is directlylabeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes are detected.

Alternatively, the reaction is conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a second, labeled antibodyspecific for the other partner to detect anchored complexes. Again,depending upon the order of addition of reactants to the liquid phase,test compounds which inhibit complex or which disrupt preformedcomplexes are identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the target polynucleotideprotein and the interacting cellular or extracellular binding partner isprepared in which either the target polynucleotide product or itsbinding partner is labeled, but the signal generated by the label isquenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 byRubenstein which utilizes this approach for immunoassays). The additionof a test substance that competes with and displaces one of the speciesfrom the preformed complex results in the generation of a signal abovebackground. In this way, test substances which disrupt targetpolynucleotide protein/cellular or extracellular binding partnerinteraction are identified.

In a particular embodiment the target polynucleotide product is preparedfor immobilization using recombinant DNA techniques described above. Forexample, the target polynucleotide coding region is fused to aglutathione-5-transferase (GST) polynucleotide using a fusion vector,such as pGEX-5X-1, in such a manner that its binding activity ismaintained in the resulting fusion protein. The interactive cellular orextracellular binding partner is purified and used to raise a monoclonalantibody, using methods routinely practiced in the art and as describedbelow. This antibody is labeled with the radioactive isotope “′”, forexample, by methods routinely practiced in the art. In a heterogeneousassay, e.g., the GST-target polynucleotide fusion protein is anchored toglutathione-agarose beads. The interactive cellular or extracellularbinding partner is then added in the presence or absence of the testcompound in a manner that allows interaction and binding to occur. Atthe end of the reaction period, unbound material can be washed away, andthe labeled monoclonal antibody is added to the system and allowed tobind to the complexed components. The interaction between the targetpolynucleotide protein and the interactive cellular or extracellularbinding partner is detected by measuring the amount of radioactivitythat remains associated with the glutathione-agarose beads. A successfulinhibition of the interaction by the test compound results in a decreasein measured radioactivity.

Alternatively, the GST-target polynucleotide fusion protein and theinteractive cellular or extracellular binding partner are mixed togetherin liquid in the absence of the solid glutathione-agarose beads. Thetest compound is added either during or after the species are allowed tointeract. This mixture is added to the glutathione-agarose beads andunbound material is washed away. Again the extent of inhibition of thetarget polynucleotide product/binding partner interaction is detected byadding the labeled antibody and measuring the radioactivity associatedwith the beads.

In another embodiment of the invention, these same techniques areemployed using peptide fragments that correspond to the binding domainsof the target polynucleotide product and/or the interactive cellular orextracellular binding partner (in cases where the binding partner is aprotein), in place of one or both of the full length proteins. Anynumber of methods routinely practiced in the art are used to identifyand isolate the binding sites. These methods include, but are notlimited to, mutapolynucleotidesis of the polynucleotide encoding one ofthe proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in thepolynucleotide encoding the second species in the complex are thenselected. Sequence analysis of the polynucleotides encoding therespective proteins reveals the mutations that correspond to the regionof the protein involved in interactive binding. Alternatively, oneprotein is anchored to a solid surface using methods described above,and allowed to interact with and bind to its labeled binding partner,which has been treated with a proteolytic enzyme, such as trypsin. Afterwashing, a short, labeled peptide comprising the binding domain remainsassociated with the solid material, and can be isolated and identifiedby amino acid sequencing. Also, once the polynucleotide coding for thecellular or extracellular binding partner is obtained, shortpolynucleotide segments are engineered to express peptide fragments ofthe protein, which are tested for binding activity and purified orsynthesized.

For example, and not by way of limitation, a target polynucleotideproduct is anchored to a solid material as described, above, by making aGST-target polynucleotide fusion protein and allowing it to bind toglutathione agarose beads. The interactive cellular or extracellularbinding partner is labeled with a radioactive isotope, and cleaved witha proteolytic enzyme such as trypsin. Cleavage products are added to theanchored GST-target polynucleotide fusion protein and allowed to bind.After washing away unbound peptides, labeled bound material,representing the cellular or extracellular binding partner bindingdomain, is eluted, purified, and analyzed for amino acid sequence bywell-known methods. Peptides so identified are produced synthetically orfused to appropriate facilitative proteins using well-known recombinantDNA technology.

In one embodiment of the present invention, the proteins encoded by thefungal polynucleotides identified using the methods of the presentinvention are isolated and expressed. These recombinant proteins arethen used as targets in assays to screen libraries of compounds forpotential drug candidates. The generation of chemical libraries iswell-known in the art. For example, combinatorial chemistry is used togenerate a library of compounds to be screened in the assays describedherein. A combinatorial chemical library is a collection of diversechemical compounds generated by either chemical synthesis or biologicalsynthesis by combining a number of chemical “building block” reagents.For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining amino acids in every possiblecombination to yield peptides of a given length. Millions of chemicalcompounds theoretically can be synthesized through such combinatorialmixings of chemical building blocks. For example, one commentatorobserved that the systematic, combinatorial mixing of interchangeablechemical building blocks results in the theoretical synthesis of milliontetrameric compounds or billion pentameric compounds. (Gallop et al.,“Applications of Combinatorial Technologies to Drug Discovery,Background and Peptide Combinatorial Libraries,” Journal of MedicinalChemistry, Vol. 37, No. 9, 1233-1250 (1994). Other chemical librariesknown to those in the art may also be used, including natural productlibraries.

Once generated, combinatorial libraries are screened for compounds thatpossess desirable biological properties. For example, compounds whichmay be useful as drugs or to develop drugs would likely have the abilityto bind to the target protein identified, expressed and purified asdiscussed above. Further, if the identified target protein is an enzyme,candidate compounds would likely interfere with the enzymatic propertiesof the target protein. For example, the enzymatic function of a targetprotein may be to serve as a protease, nuclease, phosphatase,dehydrogenase, transporter protein, transcriptional enzyme, replicationcomponent, and any other type of enzyme known or unknown. Thus, thepresent invention contemplates using the protein products describedabove to screen combinatorial chemical libraries.

In some embodiments of the present invention, the biochemical activityof the protein, as well as the chemical structure of a substrate onwhich the protein acts is known. In other embodiments of the presentinvention, the biochemical activity of the target protein is unknown andthe target protein has no known substrates.

In some embodiments of the present invention, libraries of compounds areto identify compounds that function as inhibitors of the targetpolynucleotide product. First, a library of small molecules is generatedusing methods of combinatorial library formation well-known in the art.U.S. Pat. Nos. 5,463,564 and 5,574,656, to Agraflotis, et al., entitled“System and Method of Automatically Generating Chemical Compounds withDesired Properties”, the disclosures of which are incorporated herein byreference in their entireties, are two such teachings. Then the librarycompounds are screened to identify those compounds that possess desiredstructural and functional properties. U.S. Pat. No. 5,684,71, thedisclosure of which is incorporated herein by reference in its entirety,also discusses a method for screening libraries.

To illustrate the screening process, the target polynucleotide product,an enzyme, and chemical compounds of the library are combined andpermitted to interact with one another. A labeled substrate is added tothe incubation. The label on the substrate is such that a detectablesignal is emitted from metabolized substrate molecules. The emission ofthis signal permits one to measure the effect of the combinatoriallibrary compounds on the enzymatic activity of target enzymes bycomparing it to the signal emitted in the absence of combinatoriallibrary compounds. The characteristics of each library compound areencoded so that compounds demonstrating activity against the enzyme canbe analyzed and features common to the various compounds identified canbe isolated and combined into future iterations of libraries.

Once a library of compounds is screened, subsequent libraries aregenerated using those chemical building blocks that possess the featuresshown in the first round of screen to have activity against the targetenzyme. Using this method, subsequent iterations of candidate compoundswill possess more and more of those structural and functional featuresrequired to inhibit the function of the target enzyme, until a group ofenzyme inhibitors with high specificity or the enzyme can be found.These compounds can then be further tested for their safety and efficacyas antibiotics for use in mammals.

It will be readily appreciated that this particular screeningmethodology is exemplary only. Other methods are well-known to thoseskilled in the art. For example, a wide variety of screening techniquesare known for a large number of naturally-occurring targets when thebiochemical function of the target protein is known. For example, sometechniques involve the generation and use of small peptides to probe andanalyze target proteins both biochemically and genetically in order toidentify and develop drug leads. Such techniques include the methodsdescribed in PCT Publications Nos. WO9935494 and WO9819162.

Drug Screening

The fungal essential polypeptides and/or peptides of the presentinvention, or immunogenic fragments or oligopeptides thereof, can beused for screening therapeutic drugs or compounds in a variety of drugscreening techniques. The fragment employed in such a screening assaymay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The reduction or abolition ofactivity of the formation of binding complexes between the ion channelprotein and the agent being tested can be measured. Thus, the presentinvention provides a method for screening or assessing a plurality ofcompounds for their specific binding affinity with a fungal essentialpolypeptide, or a bindable peptide fragment, of this invention,comprising providing a plurality of compounds, combining the fungalessential polypeptide, or a bindable peptide fragment, with each of aplurality of compounds for a time sufficient to allow binding undersuitable conditions and detecting binding of the fungal essentialpolypeptide or peptide to each of the plurality of test compounds,thereby identifying the compounds that specifically bind to the fungalessential polypeptide or peptide.

Methods of identifying compounds that modulate the activity of the novelfungal essential polypeptides and/or peptides are provided by thepresent invention and comprise combining a potential or candidatecompound or drug modulator of biological activity with an fungalessential polypeptide or peptide, for example, the fungal essentialamino acid sequence as set forth in SEQ ID NO:2, and measuring an effectof the candidate compound or drug modulator on the biological activityof the fungal essential polypeptide or peptide. Such measurable effectsinclude, for example, physical binding interaction; the ability tocleave a suitable substrate; effects on native and cloned fungalessential-expressing cell line; and effects of modulators orother—mediated physiological measures.

Another method of identifying compounds that modulate the biologicalactivity of the novel fungal essential polypeptides of the presentinvention comprises combining a potential or candidate compound or drugmodulator of a biological activity with a host cell that expresses thefungal essential polypeptide and measuring an effect of the candidatecompound or drug modulator on the biological activity of the fungalessential polypeptide. The host cell can also be capable of beinginduced to express the fungal essential polypeptide, e.g., via inducibleexpression. Physiological effects of a given modulator candidate on thefungal essential polypeptide can also be measured. Thus, cellular assaysfor particular modulators may be either direct measurement orquantification of the physical biological activity of the fungalessential polypeptide, or they may be measurement or quantification of aphysiological effect. Such methods preferably employ a fungal essentialpolypeptide as described herein, or an overexpressed recombinant fungalessential polypeptide in suitable host cells containing an expressionvector as described herein, wherein the fungal essential polypeptide isexpressed, overexpressed, or undergoes upregulated expression.

Another aspect of the present invention embraces a method of screeningfor a compound that is capable of modulating the biological activity ofa fungal essential polypeptide, comprising providing a host cellcontaining an expression vector harboring a nucleic acid sequenceencoding a fungal essential polypeptide, or a functional peptide orportion thereof (e.g., SEQ ID NOS:2); determining the biologicalactivity of the expressed fungal essential polypeptide in the absence ofa modulator compound; contacting the cell with the modulator compoundand determining the biological activity of the expressed fungalessential polypeptide in the presence of the modulator compound. In sucha method, a difference between the activity of the fungal essentialpolypeptide in the presence of the modulator compound and in the absenceof the modulator compound indicates a modulating effect of the compound.

Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as modulators can be any small chemical compound, orbiological entity (e.g., protein, sugar, nucleic acid, lipid). Testcompounds will typically be small chemical molecules and peptides.Generally, the compounds used as potential modulators can be dissolvedin aqueous or organic (e.g., DMSO-based) solutions. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source. Assays aretypically run in parallel, for example, in microtiter formats onmicrotiter plates in robotic assays. There are many suppliers ofchemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs, Switzerland), for example. Also, compounds may besynthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned forthe detection of modulators of the novel fungal essentialpolynucleotides and polypeptides described herein. Such high throughputscreening methods typically involve providing a combinatorial chemicalor peptide library containing a large number of potential therapeuticcompounds (e.g., ligand or modulator compounds). Such combinatorialchemical libraries or ligand libraries are then screened in one or moreassays to identify those library members (e.g., particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds so identified can serve as conventional lead compounds, orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptoids(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No.5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.;Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City,Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a largenumber of combinatorial libraries are commercially available (e.g.,ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St.Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton,Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressingan ion channel is attached to a solid phase substrate. In such highthroughput assays, it is possible to screen up to several thousanddifferent modulators or ligands in a single day. In particular, eachwell of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000 -20,000 different compounds arepossible using the described integrated systems.

In another of its aspects, the present invention encompasses screeningand small molecule (e.g., drug) detection assays which involve thedetection or identification of small molecules that can bind to a givenprotein, i.e., a fungal essential polypeptide or peptide. Particularlypreferred are assays suitable for high throughput screeningmethodologies.

In such binding-based detection, identification, or screening assays, afunctional assay is not typically required. All that is needed is atarget protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shiftassay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

To purify a fungal essential polypeptide or peptide to measure abiological binding or ligand binding activity, the source may be a wholecell lysate that can be prepared by successive freeze-thaw cycles (e.g.,one to three) in the presence of standard protease inhibitors. Thefungal essential polypeptide may be partially or completely purified bystandard protein purification methods, e.g., affinity chromatographyusing specific antibody described infra, or by ligands specific for anepitope tag engineered into the recombinant fungal essential polypeptidemolecule, also as described herein. Binding activity can then bemeasured as described.

Compounds which are identified according to the methods provided herein,and which modulate or regulate the biological activity or physiology ofthe fungal essential polypeptides according to the present invention area preferred embodiment of this invention. It is contemplated that suchmodulatory compounds may be employed in treatment and therapeuticmethods for treating a condition that is mediated by the novel fungalessential polypeptides by administering to an individual in need of suchtreatment a therapeutically effective amount of the compound identifiedby the methods described herein.

In addition, the present invention provides methods for treating anindividual in need of such treatment for a disease, disorder, orcondition that is mediated by the fungal essential polypeptides of theinvention, comprising administering to the individual a therapeuticallyeffective amount of the fungal essential-modulating compound identifiedby a method provided herein.

Antibodies

Described herein are methods for the production of antibodies capable ofspecifically recognizing epitopes of one or more of the essentialpolynucleotide polypeptides described above.

On one embodiment, the antibodies of the present invention are humanantibodies capable of neutralizing a fungal pathogen in a human host,such that the human host can effectively combat the invading pathogen,and thus treat or ameliorate the symptoms caused by the invadingpathogen.

In the present invention, “epitopes” refer to polypeptide fragmentshaving antigenic or immunogenic activity in an animal. A preferredembodiment of the present invention relates to a polypeptide fragmentincluding an epitope, as well as the polynucleotide encoding thisfragment. A region of a protein molecule to which an antibody can bindis defined as an “antigenic epitope”. In contrast, an “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse. (See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA81:3998-4002 (1983).) In the present invention, antigenic epitopespreferably contain a sequence of at least six, more preferably at leastnine, and most preferably between about 15 to about 30 amino acids.Antigenic epitopes are useful to raise antibodies, including monoclonalantibodies, that specifically bind the epitope. (See, for instance,Wilson et al., Cell 37:767-778 (1984); Sutcliffe, J. G. et al., Science219:660-666 (1983)). Similarly, immunogenic epitopes can be used toinduce antibodies according to methods well-known in the art. (See, forinstance, Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; andBittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985), both of whichare herein incorporated by reference.) The immunogenic epitopes may bepresented together with a carrier protein, such as an albumin, to ananimal system (such as rabbit or mouse) or, if it is long enough (atleast about 25 amino acids), without a carrier.

Accordingly, the invention provides a method of eliciting an immuneresponse in an animal, comprising introducing into the animal animmunogenic composition comprising an isolated polypeptide, the aminoacid sequence of which comprises at least 6 consecutive residues of oneof SEQ ID NO: 12 to SEQ ID NO: 22 or one of SEQ ID NO: 48 to SEQ ID NO:73.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fefragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983).). Thus, these fragmentsare preferred, as well as the products of a FAB or other immunoglobulinexpression library.

As described above, the antibodies are preferably monoclonal, but mayalso be polyclonal. Monoclonal antibodies may be produced by methodsknown in the art. These methods include the immunological methoddescribed by Kohler and Millstein in Nature vol. 256, pp 495-497 (1975)and by Campbell in “Monoclonal Antibody Technology, The Production AndCharacterization Of Rodent And Human Hybridomas,” in Burdon, et al.(Eds.), Laboratory Techniques in Biochemistry and Molecular Biology,vol. 13, Elsebier Science Publishers, Amsterdam, NE (1985); and Coligan,J. E., et al. (Eds.), Current Protocols in Immunology, WileyIntersciences, Inc., New York, (1999); as well as the recombinant DNAmethod described by Huse, et al., Science, 246:1275-1281 (1989). Therecombinant DNA method preferably comprises screening phage librariesfor human antibody fragments.

In order to produce monoclonal antibodies, a host mammal is inoculatedwith an essential polynucleotide peptide or peptide fragment asdescribed above, and then boosted. Spleens are collected from inoculatedmammals a few days after the final boost. Cell suspensions from thespleens are fused with a target cell in accordance with the generalmethod described by Kohler and Millstein, Nature, 256:495-497 (1975). Inorder to be useful, the peptide fragment must contain sufficient aminoacid residues to define the epitope of the molecule being detected.

Antibodies directed against an essential polynucleotide polypeptide orfragment thereof can be used therapeutically to treat an infectiousdisease by preventing infection and/or inhibiting growth of thepathogen. Antibodies can also be used to modify a biological activity ofan essential polynucleotide polypeptide. Antibodies to essentialpolynucleotide polypeptides can also be used to alleviate one or moresymptoms associated with infection by the organism. To facilitate orenhance its therapeutic effect, an antibody (or fragment thereof) may beconjugated to a therapeutic moiety such as a toxin or fungicidal agent.Techniques for conjugating a therapeutic moiety to antibodies arewell-known, see, e.g., Thorpe et al., Immunol. Rev., 62: 119-58 (1982).

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:12 to 22, and/or anepitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, monovalent, bispecific, heteroconjugate, multispecific,human, humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. The term “antibody,” as used herein,refers to immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds an antigen. Theimmunoglobulin molecules of the invention can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule. Moreover, the term“antibody” (Ab) or “monoclonal antibody” (Mab) is meant to includeintact molecules, as well as, antibody fragments (such as, for example,Fab and F(ab′)2 fragments) which are capable of specifically binding toprotein. Fab and F(ab′)2 fragments lack the Fc fragment of intactantibody, clear more rapidly from the circulation of the animal orplant, and may have less non-specific tissue binding than an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, thesefragments are preferred, as well as the products of a FAB or otherimmunoglobulin expression library. Moreover, antibodies of the presentinvention include chimeric, single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homologue of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologues of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M,5×10-6 M, 10-6M, 5×10-7 M, 107 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M,5×10-10 M, 10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M,10-13 M, 5×10-14 M, 10-14 M, 5×10-15 M, or 10-15 M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionucleotides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art.

The antibodies of the present invention may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Coldspring Harbor Laboratory Press, 2^(nd) ed. (1988); and CurrentProtocols, Chapter 2; which are hereby incorporated herein by referencein its entirety). In a preferred method, a preparation of the fungalessential polynucleotide protein is prepared and purified to render itsubstantially free of natural contaminants. Such a preparation is thenintroduced into an animal in order to produce polyclonal antisera ofgreater specific activity. For example, a polypeptide of the inventioncan be administered to various host animals including, but not limitedto, rabbits, mice, rats, etc. to induce the production of seracontaining polyclonal antibodies specific for the antigen. Theadministration of the polypeptides of the present invention may entailone or more injections of an immunizing agent and, if desired, anadjuvant. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, and include but are not limitedto, Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum. Suchadjuvants are also well known in the art. For the purposes of theinvention, “immunizing agent” may be defined as a polypeptide of theinvention, including fragments, variants, and/or derivatives thereof, inaddition to fusions with heterologous polypeptides and other forms ofthe polypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections, thoughthey may also be given intramuscularly, and/or through IV). Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivitizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The antibodies of the present invention may comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., pp. 563-681 (1981); Köhler et al., Eur. J.Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies includes, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a humanimmune system, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The immunizing agent will typically include polypeptides of the presentinvention or a fusion protein thereof. Preferably, the immunizing agentconsists of an fungal essential polynucleotide polypeptide or, morepreferably, with a fungal essential polynucleotidepolypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56 degrees C.), and supplemented with about 10 g/lof nonessential amino acids, about 1,000 U/ml of penicillin, and about100 ug/ml of streptomycin. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986), pp. 59-103). Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. More preferred are the parent myeloma cellline (SP20) as provided by the ATCC. As inferred throughout thespecification, human myeloma and mouse-human heteromyeloma cell linesalso have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptides of the present invention. Preferably, the bindingspecificity of monoclonal antibodies produced by the hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay(ELISA). Such techniques are known in the art and within the skill ofthe artisan. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra, and/or according to Wands et al. (Gastroenterology80:225-232 (1981)). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-sepharose, hydroxyapatite chromatography, gel exclusionchromatography, gel electrophoresis, dialysis, or affinitychromatography.

The skilled artisan would acknowledge that a variety of methods exist inthe art for the production of monoclonal antibodies and thus, theinvention is not limited to their sole production in hydridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to polynucleotidesencoding the heavy and light chains of murine antibodies, or such chainsfrom human, humanized, or other sources). The hydridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transformedinto host cells such as Simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison et al, supra) or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of theinvention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fe region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples described herein. In a non-limitingexample, mice can be immunized with a polypeptide of the invention or acell expressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagepolynucleotide III or polynucleotide VIII protein. Examples of phagedisplay methods that can be used to make the antibodies of the presentinvention include those disclosed in Brinkman et al., J. Immunol.Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persicet al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabillyet al., Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985);U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which areincorporated herein by reference in their entirety. Humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and a framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the methods ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin (Jones etal., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of cole et al., andBoerder et al., are also available for the preparation of humanmonoclonal antibodies (cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(1):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin polynucleotides. For example, the humanheavy and light chain immunoglobulin polynucleotide complexes may beintroduced randomly or by homologous recombination into mouse embryonicstem cells. Alternatively, the human variable region, constant region,and diversity region may be introduced into mouse embryonic stem cellsin addition to the human heavy and light chain polynucleotides. Themouse heavy and light chain immunoglobulin polynucleotides may berendered non-functional separately or simultaneously with theintroduction of human immunoglobulin loci by homologous recombination.In particular, homozygous deletion of the JH region prevents endogenousantibody production. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, e.g., all or a portion of a polypeptide of theinvention. Monoclonal antibodies directed against the antigen can beobtained from the immunized, transgenic mice using conventionalhybridoma technology. The human immunoglobulin transpolynucleotidesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol.13:65-93 (1995). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877;U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which areincorporated by reference herein in their entirety. In addition,companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose,Calif.), and Medarex, Inc. (Princeton, N.J.) can be engaged to providehuman antibodies directed against a selected antigen using technologysimilar to that described above.

Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin polynucleotides have been partially orcompletely inactivated. Upon challenge, human antibody production isobserved, which closely resembles that seen in humans in all respects,including polynucleotide rearrangement, assembly, and creation of anantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,106, and in the following scientific publications: Marks et al.,Biotechnol., 10:779-783 (1992); Lonberg et al., Nature 368:856-859(1994); Fishwild et al., Nature Biotechnol., 14:845-51 (1996);Neuberger, Nature Biotechnol., 14:826 (1996); Lonberg and Huszer,Intern. Rev. Immunol., 13:65-93 (1995).

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Such anti-idiotypic antibodies capable of binding to the fungalessential polynucleotide polypeptide can be produced in a two-stepprocedure. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodythat binds to a second antibody. In accordance with this method, proteinspecific antibodies are used to immunize an animal, preferably a mouse.The splenocytes of such an animal are then used to produce hybridomacells, and the hybridoma cells are screened to identify clones thatproduce an antibody whose ability to bind to the protein-specificantibody can be blocked by the polypeptide. Such antibodies compriseanti-idiotypic antibodies to the protein-specific antibody and can beused to immunize an animal to induce formation of furtherprotein-specific antibodies.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, Preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities maybe directed towards a polypeptide of the present invention, the othermay be for any other antigen, and preferably for a cell-surface protein,receptor, receptor subunit, tissue-specific antigen, virally derivedprotein, virally encoded envelope protein, bacterially derived protein,or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for the treatment of HIV infection (WO 91/00360; WO 92/20373; andEP03089). It is contemplated that the antibodies may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioester bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:12 to 22.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particularpolynucleotide sequence to identify, e.g., a cDNA clone from a cDNAlibrary that encodes the antibody. Amplified nucleic acids generated byPCR may then be cloned into replicable cloning vectors using any methodwell known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutapolynucleotidesis, PCR, etc. (see, for example, the techniquesdescribed in Sambrook et al., 1990, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,John Wiley & Sons, NY, which are both incorporated by reference hereinin their entireties), to generate antibodies having a different aminoacid sequence, for example to create amino acid substitutions,deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing polynucleotides from a mouse antibodymolecule of appropriate antigen specificity together withpolynucleotides from a human antibody molecule of appropriate biologicalactivity can be used. As described supra, a chimeric antibody is amolecule in which different portions are derived from different animalspecies, such as those having a variable region derived from a murinemAb and a human immunoglobulin constant region, e.g., humanizedantibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

More preferably, a clone encoding an antibody of the present inventionmay be obtained according to the method described in the Example sectionherein.

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early polynucleotide promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem . .. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned targetpolynucleotide product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign polynucleotides. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric polynucleotidemay then be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing the antibody molecule in infectedhosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359(1984)). Specific initiation signals may also be required for efficienttranslation of inserted antibody coding sequences. These signals includethe ATG initiation codon and adjacent sequences. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (see Bittner et al.,Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes thepolynucleotide product in the specific fashion desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andpolynucleotide products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the polynucleotide product may beused. Such mammalian host cells include but are not limited to CHO,VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, and in particular, breastcancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 andT47D, and normal mammary gland cell line such as, for example, CRL7030and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))polynucleotides can be employed in tk-, hgprt- or aprt-cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following polynucleotides: dhfr, which confersresistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357(1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt,which confers resistance to mycophenolic acid (Mulligan & Berg, Proc.Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance tothe aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan andAnderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH11(5):155-215); and hygro, which confers resistance to hygromycin(Santerre et al., Gene 30:147 (1984)). Methods commonly known in the artof recombinant DNA technology may be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley& Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981),which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on polynucleotide amplification for the expression ofcloned polynucleotides in mammalian cells in DNA cloning, Vol. 3.(Academic Press, New York, 1987)). When a marker in the vector systemexpressing antibody is amplifiable, increase in the level of inhibitorpresent in culture of host cell will increase the number of copies ofthe marker polynucleotide. Since the amplified region is associated withthe antibody gene, production of the antibody will also increase (Crouseet al., Mol. Cell. Biol. 3:257 (1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341 (1992) (said references incorporated by reference intheir entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:12 to 22 may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:12 to22 may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson etal., J. Biol. Chem . . . 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, I-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof. Therapeutic agents include, but are not limitedto, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

The present invention also encompasses the creation of syntheticantibodies directed against the polypeptides of the present invention.One example of synthetic antibodies is described in Radrizzani, M., etal., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class ofsynthetic antibodies has been described and are referred to asmolecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies,peptides, and enzymes are often used as molecular recognition elementsin chemical and biological sensors. However, their lack of stability andsignal transduction mechanisms limits their use as sensing devices.Molecularly imprinted polymers (MIPs) are capable of mimicking thefunction of biological receptors but with less stability constraints.Such polymers provide high sensitivity and selectivity while maintainingexcellent thermal and mechanical stability. MIPs have the ability tobind to small molecules and to target molecules such as organics andproteins' with equal or greater potency than that of natural antibodies.These “super” MIPs have higher affinities for their target and thusrequire lower concentrations for efficacious binding.

During synthesis, the MIPs are imprinted so as to have complementarysize, shape, charge and functional groups of the selected target byusing the target molecule itself (such as a polypeptide, antibody,etc.), or a substance having a very similar structure, as its “print” or“template.” MIPs can be derivatized with the same reagents afforded toantibodies. For example, fluorescent ‘super’ MIPs can be coated ontobeads or wells for use in highly sensitive separations or assays, or foruse in high throughput screening of proteins.

Moreover, MIPs based upon the structure of the polypeptide(s) of thepresent invention may be useful in screening for compounds that bind tothe polypeptide(s) of the invention. Such a MIP would serve the role ofa synthetic “receptor” by minimicking the native architecture of thepolypeptide. In fact, the ability of a MIP to serve the role of asynthetic receptor has already been demonstrated for the estrogenreceptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001);Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71,(2001)). A synthetic receptor may either be mimicked in its entirety(e.g., as the entire protein), or mimicked as a series of short peptidescorresponding to the protein (Rachkov, A., Minoura, N, Biochim, Biophys,Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may beemployed in any one or more of the screening methods described elsewhereherein.

MIPs have also been shown to be useful in “sensing” the presence of itsmimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron.,16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst.,126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst.,126(6):798-802, (2001)). For example, a MIP designed using a polypeptideof the present invention may be used in assays designed to identify, andpotentially quantitate, the level of said polypeptide in a sample. Sucha MIP may be used as a substitute for any component described in theassays, or kits, provided herein (e.g., ELISA, etc.).

A number of methods may be employed to create MIPs to a specificreceptor, ligand, polypeptide, peptide, organic molecule. Severalpreferred methods are described by Esteban et al in J. Anal, Chem.,370(7):795-802, (2001), which is hereby incorporated herein by referencein its entirety in addition to any references cited therein. Additionalmethods are known in the art and are encompassed by the presentinvention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem,Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J.,De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54,(2001); which are hereby incorporated by reference in their entiretyherein.

The antibodies of the present invention have various utilities. Forexample, such antibodies may be used in diagnostic assays to detect thepresence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), pp 147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present inventionare useful for the affinity purification of such polypeptides fromrecombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of thepolynucleotide of the present invention may be useful as a cell specificmarker, or more specifically as a cellular marker that is differentiallyexpressed at various stages of differentiation and/or maturation ofparticular cell types. Monoclonal antibodies directed against a specificepitope, or combination of epitopes, will allow for the screening ofcellular populations expressing the marker. Various techniques can beutilized using monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-2 M,10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M,10-6 M, 5×10-7 M, 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M,10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M,5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Antibodies directed against polypeptides of the present invention areuseful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the polynucleotide encoding anantibody directed against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody polynucleotide such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or polynucleotide therapy applicationsof the present invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced ina plant (e.g., cloning the polynucleotide of the antibody directedagainst a polypeptide of the present invention, and transforming a plantwith a suitable vector comprising said polynucleotide for constitutiveexpression of the antibody within the plant), and the plant subsequentlyingested by an animal, thereby conferring temporary immunity to theanimal for the specific antigen the antibody is directed towards (See,for example, U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferablypolyclonal antibodies, more preferably monoclonal antibodies, and mostpreferably single-chain antibodies, can be used as a means of inhibitingpolynucleotide expression of a particular gene, or polynucleotides, in ahuman, mammal, and/or other organism. See, for example, InternationalPublication Number WO 00/05391, published Feb. 3, 2000, to DowAgrosciences LLC. The application of such methods for the antibodies ofthe present invention are known in the art, and are more particularlydescribed elsewhere herein.

In yet another embodiment, antibodies of the present invention may beuseful for multimerizing the polypeptides of the present invention. Forexample, certain proteins may confer enhanced biological activity whenpresent in a multimeric state (i.e., such enhanced activity may be dueto the increased effective concentration of such proteins whereby moreprotein is available in a localized location).

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofpolynucleotide therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for polynucleotide therapy available in the art canbe used according to the present invention. Exemplary methods aredescribed below.

For general reviews of the methods of polynucleotide therapy, seeGoldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan andAnderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH11(5):155-215 (1993). Methods commonly known in the art of recombinantDNA technology which can be used are described in Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);and Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990).

In a preferred aspect, the compound comprises polynucleotides encodingan antibody, said polynucleotides being part of expression vectors thatexpress the antibody or fragments or chimeric proteins or heavy or lightchains thereof in a suitable host. In particular, such polynucleotideshave promoters operably linked to the antibody coding region, saidpromoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the antibody coding sequences and any otherdesired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the antibody encoding nucleic acids(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989);Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, theexpressed antibody molecule is a single chain antibody; alternatively,the polynucleotides include sequences encoding both the heavy and lightchains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo polynucleotide therapy.

In a specific embodiment, the polynucleotides are directly administeredin vivo, where it is expressed to produce the encoded product. This canbe accomplished by any of numerous methods known in the art, e.g., byconstructing them as part of an appropriate nucleic acid expressionvector and administering it so that they become intracellular, e.g., byinfection using defective or attenuated retrovirals or other viralvectors (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a polynucleotide gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering them in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol.Chem . . . 262:4429-4432 (1987)) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment,nucleic acid-ligand complexes can be formed in which the ligandcomprises a fusogenic viral peptide to disrupt endosomes, allowing thenucleic acid to avoid lysosomal degradation. In yet another embodiment,the nucleic acid can be targeted in vivo for cell specific uptake andexpression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO93/20221). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors that contains polynucleotidesencoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The polynucleotides encoding the antibody to beused in polynucleotide therapy are cloned into one or more vectors,which facilitates delivery of the polynucleotide into a patient. Moredetail about retroviral vectors can be found in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 polynucleotide to hematopoietic stem cells inorder to make the stem cells more resistant to chemotherapy. Otherreferences illustrating the use of retroviral vectors in polynucleotidetherapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in polynucleotidetherapy. Adenoviruses are especially attractive vehicles for deliveringpolynucleotides to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based polynucleotide therapy. Bout et al., Human Gene Therapy5:3-10 (1994) demonstrated the use of adenovirus vectors to transferpolynucleotides to the respiratory epithelia of rhesus monkeys. Otherinstances of the use of adenoviruses in polynucleotide therapy can befound in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al.,Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234(1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy2:775-783 (1995). In a preferred embodiment, adenovirus vectors areused.

Adeno-associated virus (AAV) has also been proposed for use inpolynucleotide therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146).

Another approach to polynucleotide therapy involves transferring apolynucleotide to cells in tissue culture by such methods aselectroporation, lipofection, calcium phosphate mediated transfection,or viral infection. Usually, the method of transfer includes thetransfer of a selectable marker to the cells. The cells are then placedunder selection to isolate those cells that have taken up and areexpressing the transferred polynucleotide. Those cells are thendelivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the polynucleotides, cell fusion, chromosome-mediatedpolynucleotide transfer, microcell-mediated polynucleotide transfer,spheroplast fusion, etc. Numerous techniques are known in the art forthe introduction of foreign polynucleotides into cells (see, e.g.,Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al.,Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m(1985) and may be used in accordance with the present invention,provided that the necessary developmental and physiological functions ofthe recipient cells are not disrupted. The technique should provide forthe stable transfer of the nucleic acid to the cell, so that the nucleicacid is expressible by the cell and preferably heritable and expressibleby its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes ofpolynucleotide therapy encompass any desired, available cell type, andinclude but are not limited to epithelial cells, endothelial cells,keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells suchas Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for polynucleotide therapy isautologous to the patient.

In an embodiment in which recombinant cells are used in polynucleotidetherapy, polynucleotides encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof polynucleotide therapy comprises an inducible promoter operablylinked to the coding region, such that expression of the nucleic acid iscontrollable by controlling the presence or absence of the appropriateinducer of transcription. Demonstration of Therapeutic or ProphylacticActivity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a polynucleotide gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (see e.g.,Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 0.1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level ofpolynucleotide expression with a standard polynucleotide expressionlevel, whereby an increase or decrease in the assayed polypeptidepolynucleotide expression level compared to the standard expressionlevel is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level ofpolynucleotide expression with a standard polynucleotide expressionlevel, whereby an increase or decrease in the assayed polypeptidepolynucleotide expression level compared to the standard expressionlevel is indicative of a particular disorder. With respect to cancer,the presence of a relatively high amount of transcript in biopsiedtissue from an individual may indicate a predisposition for thedevelopment of the disease, or may provide a means for detecting thedisease prior to the appearance of actual clinical symptoms. A moredefinitive diagnosis of this type may allow health professionals toemploy preventative measures or aggressive treatment earlier therebypreventing the development or further progression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting proteinpolynucleotide expression include immunoassays, such as the enzymelinked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).Suitable antibody assay labels are known in the art and include enzymelabels, such as, glucose oxidase; radioisotopes, such as iodine (125I,121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), andtechnetium (99Tc); luminescent labels, such as luminol; and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Antisense Molecules

The invention also provides for the identification of compounds thatmodulate (e.g., activate or inhibit) the function of a polypeptide ofthe invention. Such compounds can provide lead-compounds for developingdrugs for diagnosing and/or treating conditions associated with fungalinfections. The modulator is a compound that may alter the function of apolypeptide of the invention including SEQ ID NO: 12 through to SEQ IDNO 22, such as activating or inhibiting the function of a polypeptide ofthe invention. For example, the compound can act as an agonist,antagonist, partial agonist, partial antagonist, cytotoxic agent,inhibitor of cell proliferation, and cell proliferation-promotingagents. The activity of the compound may be known, unknown or partiallyknown.

In one embodiment, an antisense molecule is used as an antagonist of apolynucleotide product of the nucleic acid molecules of the invention.The present invention also provides the therapeutic or prophylactic useof nucleic acids of at least six nucleotides that are antisense to atarget essential polynucleotide or a portion thereof. An “antisense”target nucleic acid as used herein refers to a nucleic acid capable ofhybridizing to a portion of a target polynucleotide RNA or mRNA byvirtue of some sequence complementarity. The invention further providespharmaceutical compositions comprising an effective amount of theantisense nucleic acids of the invention in a pharmaceutical acceptablecarrier as described below.

In another embodiment, the invention is directed to methods forinhibiting the expression of a target polynucleotide in an organism ofinterest, such as C. albicans either in vitro or in vivo comprisingproviding the cell with an effective amount of a composition comprisingan antisense nucleic acid of the invention.

It is preferred that in vitro studies are first performed to quantitatethe ability of the antisense molecule to inhibit polynucleotideexpression. It is preferred that these studies utilize controls thatdistinguish between antisense polynucleotide inhibition and nonspecificbiological effects of oligonucleotides. It is also preferred that thesestudies compare levels of the target RNA or protein with that of aninternal control RNA or protein. Additionally, it is envisioned thatresults obtained using the antisense oligonucleotide are compared withthose obtained using a control oligonucleotide. It is preferred that thecontrol oligonucleotide is of approximately the same length as the testoligonucleotide and that the nucleotide sequence of the oligonucleotidediffers from the antisense sequence no more than is necessary to preventspecific hybridization to the target sequence.

Antisense molecules of the invention may be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Applied Biosystems, Palo Alto,Calif.). As examples, phosphorothioate oligonucleotides may besynthesized by the method of Steinet et al. (1988, Nucl. Acids Res.16:3209), methylphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (Sarin et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85:7448-745 1), etc.

Antisense nucleotides complementary to the coding region of a targetpolynucleotide may be used, as well as those complementary to thetranscribed untranslated region.

Antisense oligonucleotides may be single or double stranded. Doublestranded RNA's may be designed based upon the teachings of Paddison etal., Proc. Nat. Acad. Sci., 99:1443-1448 (2002); and InternationalPublication Nos. WO 01/29058, and WO 99/32619; which are herebyincorporated herein by reference.

Pharmaceutical compositions of the invention comprising an effectiveamount of an antisense nucleic acid in a pharmaceutically acceptablecarrier can be administered to a subject infected with the pathogen ofinterest.

The amount of antisense nucleic acid which will be effective in thetreatment of a particular disease caused by the fungal pathogen willdepend on the site of the infection or condition. Where possible, it isdesirable to determine the antisense cytotoxicity of the fungus to betreated in vitro, and then in useful animal model systems prior totesting and use in humans.

A number of methods have been developed for delivering antisense DNA orRNA to cells; e.g., antisense molecules can be injected directly intothe tissue site in which the pathogens are residing, or modifiedantisense molecules, designed to target the desired cells (e.g.,antisense molecule linked to peptides or antibodies that specificallybind receptors or antigens expressed on the pathogen's cell surface) canbe administered systemically. Antisense molecules can be delivered tothe desired cell population via a delivery complex. In a specificembodiment, pharmaceutical compositions comprising antisense nucleicacids of the target polynucleotides are administered via biopolymers(e.g., poly-β-1-4-N-acetylglucosamine polysaccharide), liposomes,microparticles, or microcapsules.

In various embodiments of the invention, it may be useful to use suchcompositions to achieve sustained release of the antisense nucleicacids. In a specific embodiment, it may be desirable to utilizeliposomes targeted via antibodies to specific identifiable pathogenantigens (Leonetti et al., 1990, Proc. Nat. Acad. Sci. U.S.A.87:2448-2451; Renneisen et al., 1990, J. Biol. Chem. 265:16337-16342).

Transcriptional Profiling

Gene expression profiling techniques are important tools for theidentification of antifungal compounds. To carry out profiling,polynucleotide expression arrays and microarrays can be employed. Geneexpression arrays are high density arrays of DNA samples deposited atspecific locations on, for example, a glass surface or nylon membrane.Such arrays are used by researchers to quantify relative polynucleotideexpression under different conditions. An example of this technology isfound in U.S. Pat. No. 5,807,522, which is hereby incorporated byreference.

It is possible to study the expression of substantially all of thepolynucleotides in the genome of a particular microbial organism using asingle array. For example, the arrays may consist of 12×24 cm nylonfilters containing a PCR product corresponding to ORFs from Candidaalbicans. 10 ng of each PCR product may be spotted for example every 1.5mm on the filter. Single stranded labeled cDNAs are prepared forhybridization to the array and placed in contact with the filter. Thus,the labeled cDNAs are of “antisense” orientation. Quantitative analysismay be done using a phosphorimager.

Hybridization of cDNA made from a sample of total cellular mRNA to suchan array followed by detection of binding by one or more of varioustechniques known to those in the art provides a signal at each locationon the array to which cDNA is hybridized. The intensity of thehybridization signal obtained at each location in the array thusreflects the amount of mRNA for that specific polynucleotide that waspresent in the sample. Comparing the results obtained for mRNA isolatedfrom cells grown under different conditions thus allows for a comparisonof the relative amount of expression of each individual polynucleotidesduring growth under different conditions.

Gene expression arrays are use to analyze the total mRNA expressionpattern at various time points after reduction in the level or activityof a polynucleotide product required for fungal proliferation. Reductionof the level or activity of the polynucleotide product is accomplishedby growing a diploid strain of the invention under conditions in whichthe product of the nucleic acid linked to the MET3 promoter is ratelimiting for fungal growth or survival or proliferation or by contactingthe cells with an agent which reduces the level or activity of thetarget polynucleotide product. Analysis of the expression patternindicated by hybridization to the array provides information on otherpolynucleotides whose expression is influenced by reduction in the levelor activity of the polynucleotide product. For example, levels of othermRNAs may be observed to increase, decrease or stay the same followingreduction in the level or activity of the polynucleotide productrequired for growth survival or proliferation. Thus, the mRNA expressionpattern observed following reduction in the level or activity of apolynucleotide product required for growth, survival or proliferationidentifies other nucleic acids required for expression patterns observedwhen the fungi are exposed to candidate drug compounds or knownantibiotics are compared to those observed when the level or activity ofa polynucleotide product required for fungal growth survival orproliferation is reduced. If the mRNA expression pattern observed withthe candidate drug compound is similar to that observed with the levelof the polynucleotide product is reduced, the drug compound is apromising therapeutic candidate. The assay is useful in assisting in theselection of promising candidate drug compounds for use in drugdevelopment.

In another embodiment, the present invention provides a method ofquantitative analysis of the expressed protein complement of a diploidfungal cell: a first protein expression profile is developed for acontrol diploid fungus, which has two unmodified copies of the targetpolynucleotide. Mutants of the control strain, in which one copy of thetarget polynucleotide is inactivated, for example, one of the strains ofthe present invention, by insertion of disruption cassette is generated.The allele is modified such that expression of the allele is under thecontrol of a MET3 promoter. A second protein expression profile isdeveloped for this mutant fungus under conditions where the secondallele is substantially overexpressed as compared to the expression ofthe two alleles of the polynucleotide in the control strain. Similarly,if desired, a third protein expression profile is developed underconditions where the second allele is substantially underexpressed ascompared to the expression of the two alleles of the polynucleotide inthe control strain. The first protein expression profile is thencompared with the second expression profile and if applicable to athird, forth, fifth or sixth or more expression profile to identify anexpressed protein detected a higher level in the second profile and ifapplicable at a lower level in the third profile, etc., as compared tothe level in the first profile.

Accordingly, the invention provides a method for evaluating a compoundagainst a target polynucleotide product encoded by a nucleotide sequencecomprising one of SEQ ID NO 1 to 11 said method comprising the steps of(a) contacting wild type diploid fungal cells or control cells with thecompound and generating a first protein expression profile; (b)determining the protein expression profile of mutant diploid fungalcells such as a strain of the invention which have been cultured underconditions wherein the second allele of the target polynucleotide issubstantially underexpressed not expressed or overexpressed andgenerating a second protein expression profile for the cultured cellsand comparing the first protein expression profile with the secondprotein expression profile to identify similarity in profiles. Forcomparisons, similarities of profiles can be expressed as an indicatorvalue; and the higher the indicator value, the more desirable is thecompound.

The pattern of expression of a set of proteins in a strain of theinvention may be determined by methods well-known in the art forestablishing a protein expression pattern such as two-dimensional gelelectrophoresis. A plurality of protein expression patterns will begenerated for a strain of the invention when the strain is culturedunder different conditions and different levels of expression of one ofthe modified alleles.

Pharmaceutical Compositions and Uses Thereof

Compounds including nucleic acid molecules that are identified by themethods of the invention as described herein can be administered to asubject at therapeutically effective doses to treat or preventinfections by a fungal organism such as Candia albicans. Atherapeutically effective dose refers to that amount of a compound(including nucleic acid molecules) sufficient to result in a healthfulbenefit in the treated subject. Typically, but not so limited, thecompounds act by reducing the activity or level of polynucleotideproduct encoded by a nucleic acid comprising a sequence selected fromthe group consisting of SEQ ID NO 1 to SEQ ID NO: 11, or homologuesthereof.

To treat a patient afflicted with a fungal infection it may bebeneficial to deliver an essential polynucleotide polypeptide,polynucleotide or modulating agent to the intracellular space. Suchtargeting may be achieved using well-known techniques, such as throughthe use of polyethylene glycol or liposomes, as described in Turrens,Xenobiotica 21:103 3-1040 (1991), herein incorporated by reference.

For certain embodiments, it may be beneficial to also link a drug to apolypeptide or modulating agent. As used herein, the term “drug” refersto any bioactive agent intended for administration to a mammal toprevent or treat an undesirable condition.

To prepare a pharmaceutical composition, an effective amount of one ormore polypeptides, polynucleotides and/or modulating agents is mixedwith a suitable pharmaceutical carrier. Solutions or suspensions usedfor parenteral, intradermal, subcutaneous or topical application caninclude, for example, a sterile diluent (such as water), salinesolution, fixed oil, polyethylene glycol, glycerin, propylene glycol orother synthetic solvent; antimicrobial agents (such as benzyl alcoholand methylparabens); antioxidants (such as ascorbic acid and sodiumbisulfite) and chelating agents (such as ethylenediaminetetraacetic acid(EDTA)); buffers (such as acetates, citrates and phosphates). Ifadministered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, polypropylene glycol and mixtures thereof. In addition, otherpharmaceutically active ingredients and/or suitable excipients such assalts, buffers and stabilizers may, but need not, be present within thecomposition.

A pharmaceutical composition is generally formulated and administered toexert a therapeutically useful effect while minimizing undesirable sideeffects. The number and degree of acceptable side effects depend uponthe condition for which the composition is administered. For example,certain toxic and undesirable side effects that are tolerated whentreating life-threatening illnesses, such as tumors, would not betolerated when treating disorders of lesser consequence. Theconcentration of active component in the composition will depend onabsorption, inactivation and excretion rates thereof, the dosageschedule and the amount administered, as well as other factors that maybe readily determined by those of skill in the art.

A polypeptide, polynucleotide or modulating agent may be prepared withcarriers that protect it against rapid elimination from the body, suchas time release formulations or coatings. Such carriers includecontrolled release formulations such as, but not limited to, implantsand microencapsulated delivery systems, and biodegradable, biocompatiblepolymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, polyorthoesters, polylactic acid and others known to those ofordinary skill in the art. Such formulations may generally be preparedusing well-known technology and administered by, for example, oral,rectal or subcutaneous implantation, or by implantation at the desiredtarget site. Sustained-release formulations may contain apolynucleotide, polypeptide or modulating agent dispersed in a carriermatrix and/or contained within a reservoir surrounded by a ratecontrolling membrane. Preferably the formulation provides a relativelyconstant level of modulating agent release. The amount of activecomponent contained within a sustained release formulation depends uponthe site of implantation, the rate and expected duration of release andthe nature of the condition to be treated or prevented.

Administration may be effected by incubation of cells ex vivo or invivo, such as by topical treatment, delivery by specific carrier or byvascular supply. Appropriate dosages and a suitable duration andfrequency of administration will be determined by such factors as thecondition of the patient, the type and severity of the patient's diseaseand the method of administration. In general, an appropriate dosage andtreatment regimen provides the polypeptide, polynucleotide and/ormodulating agent(s) in an amount sufficient to provide therapeuticand/or prophylactic benefit (i.e., an amount that ameliorates thesymptoms or treats or delays or prevents progression of the condition).

The precise dosage and duration of treatment is a function of thecondition being treated and may be determined empirically using knowntesting protocols or by testing the compositions in model systems knownin the art and extrapolating therefrom. Dosages may also vary with theseverity of the condition to be alleviated. The composition may beadministered one time, or may be divided into a number of smaller dosesto be administered at intervals of time. In general, the use of theminimum dosage that is sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using assays suitable for the condition being treated orprevented, which will be familiar to those of ordinary skill in the art,and for any particular subject, specific dosage regimens may be adjustedover time according to the individual need.

For pharmaceutical compositions comprising polynucleotides, thepolynucleotide may be present within any of a variety of deliverysystems known to those of ordinary skill in the art, including nucleicacid, bacterial and viral expression systems, and colloidal dispersionsystems such as liposomes. Appropriate nucleic acid expression systemscontain the necessary DNA sequences for expression in the patient (suchas a suitable promoter and terminating signal, as described above). TheDNA may also be “naked”, as described, for example, in Ulmer et al.,Science 259:1745-1749 (1993).

Various viral vectors that can be used to introduce a nucleic acidsequence into the targeted patient's cells include, but are not limitedto, vaccinia or other pox virus, herpes virus, retrovirus, oradenovirus. Techniques for incorporating DNA into such vectors arewell-known to those of ordinary skill in the art. Preferably, theretroviral vector is a derivative of a murine or avian retrovirusincluding, but not limited to, Moloney murine leukemia virus (MoMuLV),Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus(MuMTV), and Rous Sarcoma Virus (RSV).

A retroviral vector may additionally transfer or incorporate apolynucleotide for a selectable marker (to aid in the identification orselection of transduced cells) and/or a polynucleotide that encodes theligand for a receptor on a specific target cell (to render the vectortarget specific).

Viral vectors are typically non-pathogenic (defective), replicationcompetent viruses, which require assistance in order to produceinfectious vector particles. This assistance can be provided, forexample, by using helper cell lines that contain plasmids that encodeall of the structural polynucleotides of the retrovirus under thecontrol of regulatory sequences, but that are missing a nucleotidesequence which enables the packaging mechanism to recognize an RNAtranscript for encapsulation. Such helper cell lines include (but arenot limited to) PA317 and PA12. A retroviral vector introduced into suchcells can be packaged and vector virion produced. The vector virionsproduced by this method can then be used to infect a tissue cell line,such as NIH3T3 cells, to produce large quantities of chimeric retroviralvirions.

Another targeted delivery system for polynucleotides is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system for use as a delivery vehicle invitro and in vivo is a liposome (i.e., an artificial membrane vesicle).RNA. DNA and intact virions can be encapsulated within the aqueousinterior and delivered to cells in a biologically active form. Thepreparation and use of liposomes is well-known to those of ordinaryskill in the art.

Modulation of an essential gene-like function, either in vitro or invivo, may generally be achieved by administering a modulating agent thatinhibits essential polynucleotide transcription, translation oractivity.

Routes and frequency of administration, as well as dosage, will varyfrom individual to individual, and may be readily established usingstandard techniques.

In general, the pharmaceutical compositions may be administered byinjection (e.g., intracutaneous, intramuscular, intravenous orsubcutaneous), intranasally (e.g., by aspiration) or orally. A suitabledose is an amount of a compound that, when administered as describedabove, is capable of causing modulation of an essential gene-likeactivity that leads to an improved clinical outcome (e.g., more frequentremissions, complete or partial or longer disease-free survival) invaccinated patients as compared to non-vaccinated patients. In general,an appropriate dosage and treatment regimen provides the activecompound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. In general, suitable dose sizes willvary with the size of the patient, but will typically range from about0.1 mL to about 5 mL.

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of thepresent invention can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. polynucleotides or polypeptides, or agonists or antagonistsof the invention, can be used to treat, prevent, and/or diagnose any ofthese symptoms or diseases. In specific embodiments, polynucleotides,polypeptides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentpolynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat patients nonresponsive to one or more othercommercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, prevented, and/or diagnosed by a polynucleotideor polypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Kiebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat, prevent, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used totreat, prevent,and/or diagnose any of these symptoms or diseases. In specificembodiments, polynucleotides, polypeptides, or agonists or antagonistsof the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

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EXAMPLES Example 1 Target Identification

A concordance or whole genome parallel comparison was performed asdescribed by Bruccoleri, et al. Nucleic Acids Res 26:4482-4486 (1998) tofind polynucleotides that are conserved in both S. cervisiae and C.albicans. To generate the dataset, all C. albicans open reading frameswith an overall sequence similarity to S. cervisiae protein sequencesgreater than or equal to 40% were selected. The data were furtherrequired not to match bacterial (E. coli and B. subtilis) or humansequences at greater than 30% overall protein sequence similarity.

The sequences used herein were obtained from a variety of sources. Thesesources include the PathoGenome™ system from Genome TherapeuticsCorporation (Waltham, Mass.), GenBank, The Institute for GenomicResearch (TIGR), the Yeast Proteome Database, Proteome, Inc. Beverly,Mass, Saccharomyces Genome database, Stanford University, Stanford,Calif., the Candida albicans Sequencing Project, Stanford GenomeTechnology Center, Stanford, Calif., and the Sanger Center of theMedical Research Council of the United Kingdom. Additionally,non-microbial sequence data such as those from humans was obtained fromthe LifeSeq Database from Incyte Pharmaceuticals, Palo Alto, Calif., aswell as from public sources such as Genbank.

Where required, Incyte nucleotide sequences were translated into proteinsequences in all six possible reading frames. GTC supplied predictedprotein sequences with their data. In the case of other nucleotidesequences, the program CRITICA (Badger, J. and Olsen, G., MolecularBiology and Evolution 16: 512-524 (1999) was used. The sequences werestored in flat files on a UNIX computer system. Each predicted aminoacid sequences used was greater than 90 amino acids.

Each predicted protein sequence was compared to every other sequence (an“all-against-all”comparison). The program FASTA (Pearson, W. R. Methodsin Molecular Biology 132: 185-219 (2000)) was used for this comparison,using ktup=2 as a parameter, and all scores above the default cutoffwere stored. The output was processed and stored in a PostGres 95database. Graphical user interfaces, using web browser technology wereconstructed to query the database.

A Concordance Analysis was performed on the data. A total of 560 fungalconserved polynucleotides were identified (FCGs) of which 125polynucleotides had a known function and 435 polynucleotides had anunknown or partially known function (denoted Conserved Unknown ReadingFrames or CURFs). 49 CURFs were selected and tested for essentiality inS. cervisiae, and 11 of these were found essential in C. albicans (seebelow).

In many cases, the function of the essential polynucleotides determinedherein was suggested by the results of similarity searches. Table 2lists the putative functions of these polynucleotides encoded by SEQ IDNO: 1 through to SEQ ID NO: 11.

The putative function of the essential polypetides was determined usingcomputer-aided bioinformatic approaches including motif searching. Themotif searching approach involved using hidden Markov models (e.g.,Profile HMM, Bateman et al, Nucleic Acids Research 28: 263-266 (2000)).Global sequence similarity searches were performed using the amino acidsequences of all the conserved essential polynucleotide sequence againsta non-redundant protein database using the Smith-Waterman algorithm withdefault parameters on a TimeLogic DeCypher system (Crystal Bay, Nev.).

The percentage sequence similarity between these subsets of CURFs and S.cervisiae is listed in Table 1. Percent similarity ranged from 61% to87%. Additionally, these sequences were aligned with A. fumigatus, S.pombe and Homo sapiens. The percentage identity between the 11 essentialpolynucleotides of the invention and homologues of these speciesincluding those from S. cervisiae is listed in Table 2. Based on thisanalysis, the CURFs of the invention should be homologues topolynucleotides from additional fungal species.

TABLE 1 Similarity Between Candida albicans, CURFs and S. cervisiaeSpecies Genbank Seq Clone Sequence of Closest Accessions for ID No. IDNo. Name Homolog Closest Homolog % Similarity 12 FCG5 CaYLR100wORFSaccharomyces Z73272, S64936 75% cerevisiae 13 FCG6 CaYDR341cORFSaccharomyces S70106 79% cerevisiae 14 FCG7 CaYLR022cORF SaccharomycesZ73149, S64849 73% cerevisiae 15 FCG8 CaYOL077cORF Saccharomyces Z74819,S66770 84% cerevisiae 16 FCG10 CaYNL132wORF Saccharomyces Z46843 87%cerevisiae 17 FCG12 CaYGR145wORF Saccharomyces X85807 73% cerevisiae 18FCG13 CaYDR412wORF Saccharomyces U33007, S69697 61% cerevisiae 19 FCG14CaYOL010wORF Saccharomyces Z74752, Q08096 81% cerevisiae 20 FCG15CaYOR004wORF Saccharomyces Z74912, S61987 73% cerevisiae 21 FCG16CaYOR056cORF Saccharomyces Z70678, S66939 62% cerevisiae 22 FCG17CaYLR009wORF Saccharomyces Z73181, S64831 82% cerevisiae

Table 2 also includes the putative function and putative cellular rolebased on the annotation of the CURF homologues.

TABLE 2 Fungal CURFs Annotation, Similarity and Identity % % % %Identity Identity Identity Identity SEQ FCG Name in with with with withID # # YPD S. cerevisine A. fumigatus* S. pombe human (d2) Function*Cellular Role** 12 5 ERG27/YL 60 34, 15 31 22 3-keto sterol ErgosterolR100w reductase biosynthesis 13 6 YDR341c/ 65 54, 51 25 23 Arginine tRNAProtein synthesis RRS1 synthetase 14 7 YLR022c 56 46, 22 46 10 UnknownUnknown 15 8 YOL077c/ 70 51, 43 7 36 Unknown/required Protein synthesisBRX1 for biopolynucleotidesis of the 60S ribosomal subunit 16 10YNL132w/ 74 68, 65 62 12 Unknown (P-loop) Unknown/killer KRE33 toxinresistant 17 12 YGR145w 57 38, 31 42 8 Component of NuA3 Unknown/WDhistone domain, G-beta acetyltransferase repeat 18 13 YDR412w 43 48, 9 20 16 Unknown Unknown 19 14 YOL010w/ 68 38, 41 41 22 RNA cyclase-likeRNA RCL1 processing/ modification 20 15 YOR004w 55 41, 21 46 13 UnknownUnknown 21 16 YOR056c/ 46 43, 9  45 15 Unknown/nin one-pUnknown/associated NOB1 binding protein with the 26S proteasome 22 17YLR009w 73 46, 31 34 7 RNA-binding Protein synthesis protein/ribosome-associated *The two numbers represent percent identities derived fromalignment and query sequences, respective

FIG. 1 depicts the alignments of the top hits from a nonredundantdatabase (Bristol-Meyers Squibb, Princeton, N.J.) containing sequencesincluded from Genbank (infra). The aligned sequences of FIG. 1containing “NR” in their sequence name depict the top hit from thisdatabase. All of the top hits are S. cervisiae species. Additionally,FIG. 1 depicts the alignment between the top hit in the DERWENT patentdatabase (Alexandria, Va.). Table 3 shows the Genbank Accession No.and/or patent or patent application number in which the aligned CURFhomolog sequence van be found. As is evident from the alignment, thepercent identity for each of the 11 essential polynucleotides of theinvention is less than that from the non-redundant database hits.

TABLE 3 Sequences Aligning With CURFs of the Invention Sequence Name IDPatent No. Species PAT_PROT\BMSPATENT_ AAB94675 AAB94675 WO 200107628-A2Saccharomyces cerevisiae PAT_PROT\BMSPATENT_AAW33110 AAW33110 JP09263600-A Yeast PAT_PROT\BMSPATENT_AAB95680 AAB95680 EP1074617-A2 Homosapiens PAT_PROT\BMSPATENT_AAG46965 AAG46965 EP 1033405-A2 Arabidopsisthaliana PAT_PROT\BMSPATENT_AAB43803 AAB43803 WO 200055351-A1 Homosapiens PAT_PROT\BMSPATENT_AAB42957 AAB42957 WO 200058473-A2 Homosapiens PAT_PROT\BMSPATENT_AAB19089 AAB19089 WO 200058520-AlSaccharomyces cerevisiae PAT_PROT\BMSPATENT_AAB93917 AAB93917 EP1074617-A2 Homo sapiens PAT_PROT\BMSPATENT_AAW60075 AAW60075 WO9745535-A1 Saccharomyces cerevisiae PAT_PROT\BMSPATENT_AAB62453 AAB62453US 6221597-B1 Saccharomyces cerevisiae PAT_PROT\BMSPATENT_AAG48012AAG48012 EP 1033406-A2 Arabidopsis thaliana PAT_PROT\BMSPATENT_AAB09929AAB09929 JP 2000116383-A Homo sapiens

FIG. 2 depicts the alignments between the CURFs of the invention andsequences from A. fumigatus. As seen in Table 2, the percent identitybetween these homologues ranges from 34% to 68%.

Example 2 Strains and Growth Media Used for Identifying Essential Genes

49 CURFs were selected and tested for essentiality in S. cervisiae andthen in C. albicans. Three polynucleotides whose functions were known,ERG1, RAM2 and NMT1, were also used for verification of essentiality inC. albicans.

The strains used for this analysis included the C. albicans strainsSC5314 (wild type, BMS collection) and its derivative BWP17(ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisGarg4::hisG/arg4::hisG) obtained from A. P. Mitchell of ColumbiaUniversity. The yeast strain of S. cerevisiae used is ATCC 201390(MATa/MATα his3Δ1/his3Δ1 leu2Δ0/leu2Δ1 lys2Δ0/LYS2 met15Δ0/MET15ura3Δ0/ura30); Escherichia coli strain DH5α was used for plasmidspropagation.

Yeast extract/peptone/dextrose (YPD), synthetic complete medium (SC),and synthetic dextrose (SD) were prepared according to the standardprocedure described by Sherman, F. et al. Methods Enzymol. 184:3-21(1991). 5-Fluoroorotic acid (FOA) plates were used to select for theUra-revertant strains (Sherman, et al. (1991)). Spider (Liu, et al.,Science 266:1723-1725 (1994)) and LBC media (Lee, et al., Sabouraudia13:148-153 (1975)) were used to induce hyphal growth. Twenty percentbovine serum (Köhler, et al., Proc. Natl. Acad. Sci. 93:13223-13228(1996)) and medium 199 (Gibco BRL, Gaithersburg, Md.) were used toinduce germ tube formation. Uridine (25 μg/ml) was added according toFonzi & Irwin (Fonzi, et al., Genetics 134:717-728 (1993)) when neededto grow uridine-auxotrophic C. albicans strains. Other supplements suchas histidine and arginine were added to a concentration described bySherman, F. et al. Methods Enzymol. 184:3-21 (1991). Strains were grownat 30° C., unless otherwise noted. The storage and maintenance of C.albicans, which prevented chromosomal instability, was as previouslydescribed (Perepnikhatka, et al., J. Bacteriol. 181:4041-4049).

Example 3 Evaluation of Gene Essentiality in S. cervisiae

To evaluate polynucleotide essentiality for the homologues of thesequences encoding essential polynucleotides including SEQ ID NO: 1through to SEQ ID NO: 11 in S. cervisiae, one copy of the essentialpolynucleotides of the invention were disrupted in a diploid strainbackground. The resulting heterozygous strains were then sporulated andthe tetrads dissected to determine essentiality.

Disruption of the first allele of the polynucleotides of the inventionwas achieved via a PCR-based polynucleotide disruption approach where aPCR product containing the URA3 marker flanked by 40-50 bp ofpolynucleotide specific sequence was introduced into diploid yeast cellsto replace the wild type copy of the polynucleotide via homologousrecombination. Confirmation PCR was used to verify polynucleotidereplacement using primers designed within 100 bp upstream or downstreamof the site of crossover. A 2:2 ratio of segregation of meioticprogenies after tetrad dissection indicated that the polynucleotide ofinterest was essential for cell survival or synthetic medium. Genedisruption techniques are well documented in the literature for S.cervisiae and can be found at the web site(sequences-stanford.edu/group_deletion_project).

Example 4 PCR Based Gene Disruption in C. albicans

Gene essentiality in C. albicans was identified by two methods:PCR-based polynucleotide disruption and promoter swapping. Genedisruption was accomplished using a PCR-based polynucleotide disruptiontool (Wilson et al., J. Bacteriol. 181:1868-1874 (1999)).

The PCR-based polynucleotide disruption system used herein was purchasedfrom Dr. A. P. Mitchell of Columbia University. This system requires theuse of two markers to create homozygous disruptions in C. albicans. Atriply-marked auxotroph (Ura⁻ Arg⁻ His⁻) strain BWP17 and three sets ofplasmids -pGEM-URA3, pRS-ARG4ΔSpeI and pGEM-HIS1, each carrying a uniquemarker -URA3, ARG4 or HIS1 was used herein. A sequential disruption(from start to stop codons) of the two copies of any singlepolynucleotide can be achieved with any combination of the two selectivemarkers.

The general scheme of the approach is illustrated in FIG. 3. Basically,the scheme involves (1) design a pair of PCR primers which incorporatesequences that are able to anneal to plasmids containing markers.Examples of such marker containing plasmids include pGEM-URA3,pRS-ARSΔSpeI and pGEM-HIS1 (see FIG. 3). An example of sequences whichmay be incorporated into the primers includes 5DR, (SEQ ID NO: 221) and3DR (SEQ ID NO: 222). The forward primer additionally contains about 50to about 60 bp of flanking sequences derived from the start codon regionof a polynucleotide of interest or an open reading frame (ORF) ofinterest and this flanking sequence is attached to the 5′end of theforward (e.g., 5DR) primer. The reverse primer additionally contains50-60 bp of flanking sequences that were derived from the stop codonregion of the polynucleotide of interest or ORF of interest attached tothe 3′ end of the reverse primer (e.g., 3DR, SEQ ID NO: 222). (2) Aselective marker (e.g. URA3) is then amplified from one of the plasmidtemplates resulting in a PCR product which includes short regions ofhomology to the polynucleotide or ORF of interest on both ends thatallows for homologous recombination at the chromosomal locus whenintroduced into cells. (3) The PCR product is then used to transform thestrain (in this case BWP17) and transformants are selected that grow onan appropriate selective medium (e.g., SC-Uridine). (4) Total DNA isisolated from the transformants and the presence of the PCR constructsare verified using detection primers which are common primers used forall three plasmids. The correct construct should have its chromosomalallele replaced with the PCR fragment introduced via homologousrecombination. (5) Finally, once the first allele of the polynucleotideor ORF is disrupted, a second round of transformation with the PCRproduct amplified from a different marker (e.g., ARG4 in pRS-ARG4ΔSpeI)can be conducted to disrupt the remaining allele.

FIG. 3 depicts PCR-based polynucleotide disruption in C. albicans usingYFG1 (or your favorite polynucleotide or the polynucleotide ofinterest). As seen in FIG. 3, a first copy of YFG1 is disrupted.Disrupting the second polynucleotide copy in a heterozygous strain(YFG1/yfg1) would give rise to a null mutant that has the remaining copyof the polynucleotide disrupted (yfg1/yfg1). In the FIG, 5′-KOP and3′-KOP are a pair of polynucleotide disruption primers that are designedbased on the common primers 5DR and 3DR (see below). Confirmation PCRprimers are also indicated in the FIG and they include, in pairs,5′gene/3′-polynucleotide detecting primers, 5′gene/3′plasmid detectingprimers and 5′ plasmid detecting primer and 3′ polynucleotide detectingprimer.

Table 4 lists the SEQ ID Nos. for the primers which contain 5DR and 3DRalong with appropriate flanking sequences (under the column “PCR basedknockout primers, Mitchell's”) as well as the confirmation primers whichmay be used for this methodology. These knockout and confirmationprimers were used for both the essential polynucleotides of theinvention encoded by SEQ ID NO: 1 through to SEQ ID NO: 11 as well asthe polynucleotide encoded by SEQ ID NO: 45. Three polynucleotides whosehomologues are known to be essential in S. cervisiae, CaERG, CaRAM2 andCaNMT1 were also tested. CaAaH1 and CaNMT1 are homologues of S.cervisiae known to not be essential and these were also examined.

TABLE 4 PCR-Knockout and Gene Confirmation Primers PCR based PCR-basedknockout knockout SEQ ID Gene specific Gene specific primers primersSeq. NO of confirmation confirmation (Mitchell's) (Mitchell's) Gene NameID No. translation primers-5′) primers-3′) forward reverse) CaYLR100wORF1 12 159 160 195 196 CaYDR341cORF 2 13 161 162 197 198 CaYLR022cORF 3 14163 164 199 200 CaYOL077cORF 4 15 165 166 201 202 CaYNL132wORF 5 16 167168 203 204 CaYGR145wORF 6 17 171 172 207 208 CaYDR412wORF 7 18 173 174209 210 CaYOL010wORF 8 19 175 176 211 212 CaYOR004wORF 9 20 177 178 213214 YOR056cORF 10 21 179 180 215 216 YOR009wORF 11 22 181 182 217 218CaYJR072c — 45 169 170 205 206 CaERG — — 151 152 185 186 CaRAM2 — — 153154 187 188 CaPFY1 — — 155 156 189 190 CaNMT1 — — 157 158 191/193192/194 CaAAH1 — — 183 184 219 220

FIG. 4 depicts the results this polynucleotide disruption scheme usingCaAAH1. The knockout primers used for this assay are listed in SEQ IDNO: 219 and SEQ ID NO: 220.

Panels A and B depict the identification of a heterozygous AAH1/aah1construct. Lane M in both panels shows a 1 kb marker with sizes of someof the fragments indicated. Lane 1 in Panel A depicts a wild type strainthat did not result in any PCR products when using a 5′ primer (e.g. SEQID NO: 221) which anneals to any of the plasmids used herein (pGEM-URA3,pRS-ARSΔSpeI and pGEM-HIS1) and a 3′ primer which anneals to the 3′region of the AAH1 polynucleotide (e.g., SEQ ID NOs: 184). The wild typestrain, however seen in panel B, Lane 1, did produce the wild typepolynucleotide fragment of 0.9 kb when using 5′ and 3′ primers whichanneal to the 5′ and 3′ regions of AAH1 (e.g., SEQ ID NOs 183-184). Lane2 (Panel A) shows the construct of the heterozygous strain that gives a2.2 kb band via a common 5′ plasmid primer and a 3′polynucleotidedetecting primer (e.g., SEQ ID NOs: 221 and 184, respectively, forexample). Panel B, lane 2 shows the expected size fragments of 0.9 kband 2.3 kb when using the 5′ and 3′ polynucleotide primers.

Panel C and D show the identification of the homozygous aah1/aah1 nullmutant. Lane M is the 1 kb marker on both panels. Lane 3 contains theheterozygous strain used for obtaining the double disruptant of AAH1.Lane 4 shows the aah1/aah1 construct which correctly results in two PCRbands of 1.6 and 2.2 kb, respectively (panel C), via a 5′ common primerand a 3′ polynucleotide specific primer (primers 5′detect/3′gene) andtwo bands of 1.7 kb and 2.3 kb bands, respectively, via the 5′ and 3′polynucleotide specific primers (panel D).

Table 5 lists the results of this methodology for the essentialpolynucleotides of the invention encoded by SEQ ID NO:1 through to SEQID NO: 11, SEQ ID NO:45. Genes with known essential function were alsoexamined. These include the C. albicans homologues of ERG1, RAM2 andNMT1. The non-essential gene, PFY1 was also tested.

TABLE 5 Essential Genes Identified By PCR-Based Knockout and PromoterSwapping Cassette Essentiality Essentiality SEQ FCG via PCR- via MET3ID# # Name in YPD based KO promoter Functions — Known Genes 1 ERG1Essential Essential Squaline epoxidase — 2 RAM2/YKL019w EssentialEssential Alpha subunit of farnesyl transferase — 3 PFY1/YOR122c Not NotCell polarity essential essential — 4 NMT1/YLR195c ND* EssentialN-myristoyl transferase 12 Unknown 5 ERG27/YLR100w Essential Essential3-keto sterol polynucleotides reductase 13 or CURFs 6 YDR341c/RRS1Essential Essential Arginine tRNA synthetase 14 7 YLR022c EssentialEssential Unknown 15 8 YOL077c/BRX1 Essential Essential Unknown 16 10YNL132w/KRE33 ?** Essential Unknown (P- loop) 45 11 YJR072c EssentialNot Unknown essential 17 12 YGR145w ?** Essential Component of NuA3histone acetyltransferase 18 13 YDR412w ?** Essential Unknown 19 14YOL010w/RCL1 Essential Essential RNA processing 20 15 YOR004w EssentialEssential Unknown 21 16 YOR056c ?** Essential Protein degradation 22 17YLR009w ?** Essential Protein synthesis *Not determined. **?Essentiality couldn't be established by this methodology.

The results as shown in Table 5 show that essentiality could not beascertained using this methodology for CaYNL132w, CaYGR145w, CaYDR412w,CaYOR056c and CaYLR009w. The remainder of the polynucleotides encoded bySEQ ID NO: 1 through SEQ ID NO: 11 were determined to be essential. Asexpected, CaERG1 and CaRAM2 were found to be essential and PFY1 wasfound not to be essential. Essentiality for NMT1 was not determined.

Example 5 Construction of Met3 Promoter Plasmids

Two plasmids, pUMP and pAMP, were constructed which contain a MET3promoter cassette with one plasmid harboring the URA3 polynucleotide andthe other harboring ARG4 as the selective marker, respectively (see FIG.5 for plasmid maps). The C. albicans MET3 promoter region was amplifiedby PCR from the total DNA of strain SC5324 using the primers MET3SPHI(SEQ ID NO:222) and MET3NCOI (SEQ ID NO:224). The primers contain theSpeI and NcoI restriction sites, respectively. To construct pUMP, theMET3 promoter PCR product was cut with restriction enzymes SphI andNcoI, gel-purified and ligated to pGEM-URA3 that was linearized by SphIand NcoI. This placed the MET3 promoter sequence adjacent to URA3 yet inopposite orientation to avoid transcription read-through (FIG. 5).Construction of pAMP involved two steps. First, the C. albicans ARG4polynucleotide was released from pRS-ARG4ΔSpeI after digestion with SacIand KpnI (blunt-ended), and then ligated to the SacII (blunt-ended) andSacI sites of pGEM-URA3 thus replacing URA3. The resulting plasmid wasnamed pGEM-ARG4. Second, pGEM-ARG4 was linearized with SphII and NcoI,gel-purified and ligated with the MET3 promoter PCR product treated withSphI and NcoI, yielding plasmid pAMP.

Example 6 Promoter Swapping

Similar to the PCR-based polynucleotide disruption approach, two commonprimer sequences that allow annealing to the plasmid template weredesigned based on pUMP and pAMP. The forward common primer MET3 PF (SeqID NO:225) is derived from the same sequence as primer 3DR describedabove while the reverse common primer MET3PR (SEQ ID NO:224) is derivedfrom MET3 promoter sequence so that the primer will anneal to thesequence right in front of the ATG start codon. In order to replace theendogenous promoter of the polynucleotide of interest, the MET3 promoterswapping cassette with either URA3 or ARG4 as the selective marker isamplified from the plasmid pUMP or pAMP, respectively, using a pair ofprimers designed similarly to the ones used in the PCR-basedpolynucleotide disruption technique described above. The forward primercontains 50-60 bp of flanking sequences that are derived of sequences500-1000 bp upstream of the ATG codon of the polynucleotide of interestto ensure it would anneal upstream or on the boundary of the endogenouspromoter and this portion of the forward primer is attached to the 5′endof the forward common promoter primer MET3 PF. The reverse primer hasthe 50-60 bp of flanking sequences which are derived from the startcodon region of the polynucleotide or ORF including ATG attached to the3′ end of reverse common primer MET3PR. The resulting PCR productcontains the MET3 promoter cassette that is flanked by 50-60 bp ofsequences, on either end, homologous to the upstream promoter region andto the coding region of the polynucleotide of interest, respectively.

Once introduced into the cells heterozygous for the polynucleotide ofinterest obtained via the regular PCR-based polynucleotide disruptionapproach (supra), the MET3 promoter cassette would replace theendogenous promoter of the remaining allele via homologousrecombination.

FIG. 6 depicts this promoter swapping scheme. The first copy of YFG1, asseen in the FIG, is disrupted via the PCR-based polynucleotidedisruption approach. ARG4 is the selective marker used in this example.The native promoter of the remaining copy of YFG1 is subsequentlyreplaced by the MET3 promoter via homologous recombination. Primers usedfor confirmation PCR in pairs include 5′ promoter (i.e., apromoter-specific confirmation primer, upstream of the introduced MET3promoter)/3′-PTURA (e.g. SEQ ID NO: 114 and SEQ ID NO: 146),5′-PTURA/3′-promoter (i.e., a promoter-specific confirmation reverseprimer, downstream of the ATG start codon (see, e.g., SEQ ID NO: 147 andSEQ ID NO: 115), 5′-gene/3′ polynucleotide (e.g., if using CaERG asYFG1, SEQ ID NO: 151 and SEQ ID NO: 152). If, alternatively, URA3 isused as the selective marker and acts to disrupt the first copy of YFG1,the primer pair 5′-PTARG and 3′-PTARG will replace 5′-PTURA and3′-PTURA, respectively, for confirmation PCR (i.e., SEQ ID NO: 149 andSEQ ID NO: 150).

For example, FIG. 7 shows the results of confirmation PCR for C.albicans MET3P-ERG/erg1::ARG4 strains. Lanes labeled “M” contain 1 kbDNA markers with some fragment sizes indicated. Lanes 1 and 2 aretransformants putatively not containing the correct constructs. Lanes 3to 6 putatively contain the correct constructs (see below regardingmethionine and cysteine construct regulation). The primer pair, SEQ IDNOs 147 and 115, respectively, are used. These primers correspond to5′PTURA and sequences downstream of the ATG start codon of CaERG. Theresulting PCR was 1.6 kb (Lanes 3-6) indicating the correct construct.Similarly, the primer pair SEQ ID NOs 114, and 146 is expected to yielda 1.2 kb single band since this primer pair corresponds to a sequenceupstream of the ERG promoter and 3′-PTURA. Additionally, the FIG showsthe expected single 2.4 kb band for a correct construct resulting fromuse of the polynucleotide specific primers SEQ ID NO: 151 and SEQ ID NO:152.

Table 6 lists the SEQ ID NOs of the MET3 promoter swapping primers whichmay be used to remove the promoters associated with the essentialpolynucleotides encoded by SEQ ID NO: 1 through to SEQ ID NO: 11, SEQ IDNO: 45 and known essential and non-essential C. albicans homologues ofS. cervisiae described herein. Additionally, this table lists the SEQ IDNos. for PCR confirmation primers for promoter constructs. The 5′primers listed in this table are upstream of the introduced MET3promoter and the introduced selective marker. The 3′ primers are locateddownstream of the ATG start codon of the polynucleotide. SEQ ID NOs forpolynucleotide specific primers which may be used for confirmation todetect MET3 promoter strain constructs in Candida albicans are listed inTable 4.

TABLE 6 MET3 Promoter Swapping Primers and Promoter ConstructConfirmation Primers MET3 MET3 PCR PCR Promoter Promoter confirmationconfirmation SEQ ID Swapping Swapping primers for primers for SeqID NOof Primers Primers promoter promoter Gene Name No. translation (forward)(reverse) constructs (5′) constructs (3′) CaYLR100wORF 1 12 88 89 121122 CaYDR341cORF 2 13 90 91 123 124 CaYLR022cORF 3 14 92 93) 125 126CaYOL077cORF 4 15 94 95 127 128 CaYNL132wORF 5 16 96 97 129 130CaYGR145wORF 6 17 100 101 133 134 CaYDR412wORF 7 18 102 103 135 136CaYOL010wORF 8 19 104 105 137 138 CaYOR004wORF 9 20 106 107 139 140YOR056cORF 10 21 108 109 141 142 YOR009wORF 11 22 110 111 143 144CaYJR072c — 45 98 99 131 132 CaERG — — 80 81 114 115 RAM2 — — 82 83 116117 PFY1 — — 84 85 118 119 CaNMT1 — — 86 87 — 120 CaAAH1 — — 112 113 145146

Example 7 Further Means to Identify Met3 Promoter Constructs

MET3 promoter constructs were identified by phenotypic analysis viadown-regulation of the MET3 promoter as well as by a PCR confirmationmethod described below. Phenotypic analysis was conducted first for tworeasons. First, if the polynucleotide of interest is essential for cellgrowth, switching the MET3 promoter by adding methionine and cysteine inthe growing culture will block the cell growth yielding no growth orinhibited growth phenotype. In this way, the correct constructs areidentified without screening a large number of transformants via a PCRconfirmation test. Second, the inability to inhibit cell growth viadown-regulation of the MET3 promoter would be an early indication thatthe polynucleotide being tested might not be essential. Therefore, thefinal conclusion on whether a polynucleotide is essential depends onboth the phenotypic analysis and PCR confirmation tests.

FIG. 8 shows examples of phenotypes containing constructs resulting indown regulation of polynucleotide expression by methionine and cysteine.This figure depicts the phenotypes resulting from the incorporation ofcorrect and incorrect promoter swapping cassettes. 1/10 serial dilutionsof cells were made and 5 μl of diluted cells were spotted from left toright on SD medium (left panel) and SD supplemented with 2.5 mM ofmethionine and 0.5 mM of cysteine (right panel). The known essentialpolynucleotides are “turned off” by the addition of the amino acids whenthe correct construct was introduced into the strain after 48 hours at30° C. as evident by the lack of growth seen on the right panel. Thereis no difference in growth when the polynucleotide is non-essentialwithout regard to the incorporation of the appropriate construct.

Example 8 Drug Hypersensitivity

In order to determine if essential polynucleotide products could bereduced in fungal cells resulting in drug hypersensitivity, a highthroughput whole cell assay was designed. The known C. albicansessential polynucleotide corresponding ERG1 polynucleotide in S.cervisiae was tested. CaERG 1 codes for squaline epoxidase and is knownto be inhibited by terbinafine. The first polynucleotide copy wasdisrupted as described above and the second polynucleotide copy wasmodified by promoter swapping with MET3 promoter, also as describedabove.

The optimal cell density needed to control cell growth in a 384-wellplate within the log phase after 18 hours incubation at 35° C. was firstdetermined. For this determination, 50 ml of SD +His broth (minimalmedia with histidine) was inoculated with colonies grown on an SD platewith histidine and shaken at 35° C. overnight. Plates which containcolonies were streaked out once a week and stored on the bench at roomtemperature to eliminate any shock to the cells upon culturing. Cellswere then diluted to varying concentrations in SD +His medium. 35 μl ofthe diluted cells were dispensed into wells of a 384-well platecontaining 8 μl of 10.26% DMSO. To these wells, 10 μl of SD +His brothwas added for a final volume in ach well of 53 μl. The plate was sealedand incubated at 35° C. At different time intervals, the plate waswithdrawn from the incubator and the cell growth was determined byreading absorbance at 590 nm on a Perkin Elmer 7000 machine. 7.5×105cfu/ml was found to be the optimal stock concentration of cell densityfor inoculation of the 384 well plate within the log phase after 18hours incubation at 35° C.

In order to determine the titratability of the MET3 promoter and theoptimal methionine concentration to be used for drug sensitivity tests50 mol of SD +His broth medium was inoculated with colonies grown on anSD +His plate and shaken at 35° C. overnight. Cells were diluted fromthese cultures to 757,002 cfu/ml in SD +His medium. 35 μl of the dilutedcells were dispensed into wells of a 384-well plate containing 8 μl of10.26% DMSO. 10 μl of methionine stock solution made in SD +His brothwas added to the plates. 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125mM, 0.1563 mM, 0.078 mM, 0.039 mM and no methionine concentrations wereadded to the plate. Cell growth was monitored between 16 and 24 hours byreading the optical density at 595 nm. FIG. 9, depicts the cell growthover time with the different methionine concentrations. As is evidentfrom FIG. 9 cell growth increases with decreasing concentration ofmethionine. A 0.5 mM concentration of methionine reduces cell growth byapproximately 50%.

TABLE 7 Sensitivity of the MET3P-ERG1 Construct to Antifungal Drugs MICsμg/ml at 20 hrs Terbinafine Sordarin Fluconazole −Met +Met −Met +Met−Met +Met 4 or 8 0.5 0.5 0.5 1 1

In order to assess whether cells containing the ERG1-MET promoterconstruct would display hypersensitivity to terbinafine upon theaddition of methionine in comparison to cells for which no methioninewas added, cells were grown in the 384 well-plate as described above. Afinal concentration of 0.05 mM methinone was added to wells within oneplate of cells and a control plate, in which SD +His broth was added inlieu of methionine was added to a second plate. 8 μl of variousconcentrations of terbinafine in 10.26% DMSO was added to both thecontrol and test plates. The concentrations of terbinafine added todifferent wells ranged from 163 μg/ml to 0.02 μg/ml. FIG. 10 shows theresults of the sensitivity of MET3 promoter-CaERG1 cells to terbinafinein the absence and presence of methionine and lists the particularconcentrations of terbinafine inhibitor used. After the addition of thisinhibitor, both the control and experimental plates were incubated at35° C. After 18 hours of incubation, the OD readings were taken of theplates to measure cell growth (595 nm). FIG. 10 shows that a 4-8 foldincrease in sensitivity to the specific inhibitor terbinafine which isknown to target the CaERG1 polynucleotide product squalene epoxidasewere obtained in the presence of methionine comparing to the nomethionine control. Increased sensitivity of cells containing theERG1-MET3 was confirmed in a separate experiment using a 96-well formatplate. Table 7 shows that when the promoter activity was down regulatedby the presence of methionine in these experiments, sensitivity toterbinafine increased by 16-32 fold.

Furthermore, results of additional experiments conducted with the drugssordarin and fluconazole which are not known to be inhibitors of theCaERG1 polynucleotide product confirm that sensitivity to these drugs isnot altered by the presence of methionine.

These results establish that the MET3 promoter is titratable and targetpolynucleotide expression directed by the promoter, when down-regulated,result in cells more sensitive to specific drugs.

Example 9 High Throughput Screen

In order to test compounds on polynucleotide products efficiently andquickly, a high throughput screen was used. Using the known target ERG1under the control of the MET3 promoter and a specific inhibitor as acontrol, a high throughput screen was carried out as follows. Overnightcultures were seeded from multiple colonies grown on an SD +His plateinto 50 mls of SD +His broth. The culture was shaken overnight at 35° C.Plates which contain colonies were freshly streaked once per week andstored on the bench at room temperature to eliminate any shock to thecells upon culturing. Stock solution was prepared from the overnightculture. 0.350 mls of overnight culture were added to 14 mls of SD +Hismedia at room temperature. The stock solution is adjusted to contain1.0×10⁷ cells. The stock solution is diluted in preparation for use tocontain 757,002 cells/ml. 26,500 cells/well are plated. 35 μl of the757,002 cells/ml solution is added to each well. A 500 ml culture isused for 37 plates. For a 500 ml culture 37.85 ml of stock solution wasadded to 500 mls of SD +His at room temperature. In order to prepare theplates, 35 μl of Millipore water is added to each well in columns 1-24of the plates. 100% DMSO is added to wells in columns 21-24 using astacking multipdrop.

Preparation of QC Plates

Two plates are used as control plates. One of the control platescontains 4 ul of compounds and antibiotics in each well with known MICsin 100% DMSO. Wells also contain 162.6 μg/ml to 0 μg/ml of terbinafinetitration ranging from 162.6 μg/ml to 0 μg/ml. The blank plate contains4 μl of 100% DMSO in all wells. These are diluted as above and 8 μlstamped into daughter plates as well. SD +His media is added only tocolumns 23 and 24 using the stacking multipdrop. 35 μl of Candiaalbicans culture is added to columns 1-22 using a Multidrop on themicrobial robot. 10 μl of 2.65 mM methionine which has been diluted from250 mM in SD +His broth is added to all wells with a multidrop. Platesare placed in an incubator at 35° C. for 18 hours. Readings to assessthe amount of cell culture growth are taken at OD590 using a PerkinElmer 7000 spectrophotometer using the following additional settings:Gain 50, Integration Time 40, 3 flashes, flash delay 10, Dark # 10, TopRead, X-direction.

The screen is run using 384 well plates containing 4 μl of 100% DMSO ofa 1 mM stock in the last four columns. 25 μl of water is added tocolumns 1-24 and 8 μl of each compound is transferred to a “daughter”plate on a Cybio Cybiwell. The daughter plates used were clear tissueculture treated plates from Becton Dickinson (Bedford, Mass., catalognumber 358058).

Example 10 Method of Cloning the Fungal Curfs of the Present Invention

The fungal CURFs of the present invention may be cloned into expressionvectors for protein purification. Such vectors would be useful in thefurther elucidation of polynucleotide function, and enable theapplication of biochemical assays and the development of screeningassays either biophysically or biochemically. All 11 CURFs may beamplified by PCR from the genome of C. albicans and cloned to theGateway™ Expression System (Invitrogen, CA) for overexpression in E.coli. The Gateway™ Expression System allows for construction of either6×His-tagged or GST-tagged fusion proteins to facilitate easy and highyield protein overexpression and purification.

In brief, the cloning procedure involves three steps: 1) PCR amplifyingthe polynucleotide of interest from C. albicans genomic DNA by a primerpair of the corresponding gene; 2) cloning of PCR products via the BPreaction to the Donor vector pDONR201 to create an entry clone; 3)sequencing the cloned polynucleotide in the entry clone via PCR primerpair SeqL-A (5′-TCGCGTTAACGCTAGCATGGATCTC-3′ (SEQ ID NO:227) and SeqL-B(5′-GTAACATCAGAGATTTTGAGACAC-3′ (SEQ ID NO:228)). Once the entry cloneis made, an overexpression construct containing either 6× Histidine tagor GST-tag can be created by transferring polynucleotides from entryclones into destination vectors such as pDEST17 via the LR reaction.

The PCR reactions are carried out using Pfu Trubo™ DNA polymerase(Stragegene, La Jolla, Calif. 92037) as described in the precedingsession (session 3. DNA manipulations). As for the polynucleotidecloning procedure, the manufacture procotol as described in GATEWAY™Cloning Technology Instruction Manual may be followed essentially asdescribed (Invitrogen, Carlsbad, Calif. 92008).

Representative primer pairs for cloning the fungal CURFs of the presentinvention are provided below.

Primer Name Primer Sequence SEQ ID NO: FCG5-fpGGGGACAAGTTTGTACAAAAAAGCAGG 229 CTTGGTTCCGCGTGGTAGCATGTCACT TTTAAAGGATTCFCG5-rp GGGGACCACTTTGTACAAGAAAGCTGG 230 GTCCTAAGGTTGACGTGTATTTACTAT TTGFCG6-fp GGGGACAAGTTTGTACAAAAAAGCAGG 231 CTTGGTTCCGCGTGGTAGCATGTCAGTCGAAACAATTAG FCG6-rp GGGGACCACTTTGTACAAGAAAGCTGG 232GTCCTACATACGATTAACTGGAGTCAA AC FCG7-fp GGGGACAAGTTTGTACAAAAAAGCAGG 233CTTGGTTCCGCGTGGTAGCATGGCGGT GATTAATCAACC FCG7-rpGGGGACCACTTTGTACAAGAAAGCTGG 234 GTCCTATTCCTTTATGGCAGACATATC FCG8-fpGGGGACAAGTTTGTACAAAAAAGCAGG 235 CTTGGTTCCGCGTGGTAGCATGTCAGC TATCTATAAGGCFCG8-rp GGGGACCACTTTGTACAAGAAAGCTGG 236 GTCCTATTTAAATAAAGCATCATTGGFCG10-fp GGGGACAAGTTTGTACAAAAAAGCAGG 237 CTTGGTTCCGCGTGGTAGCATGGGTAAAAAAGCAATTGATG FCG10-rp GGGGACCACTTTGTACAAGAAAGCTGG 238GTCCTATTTTTTTGATTTCTTTGATTT C FCG12-fp GGGGACAAGTTTGTACAAAAAAGCAGG 239CTTGGTTCCGCGTGGTAGCATGGTTTT AAAATCAACAAC FCG12-rpGGGGACCACTTTGTACAAGAAAGCTGG 240 GTCCTACATACCTCTAAACTTATTCTT G FCG13-fpGGGGACAAGTTTGTACAAAAAAGCAGG 241 CTTGGTTCCGCGTGGTAGCATGGCAGGATTTAAAAAGAATAG FCG13-rp GGGGACCACTTTGTACAAGAAAGCTGG 242GTCCTACTTCTTGCCCTTTGATTTTG FCG14-fp GGGGACAAGTTTGTACAAAAAAGCAGG 243CTTGGTTCCGCGTGGTAGCATGTCCAG TGTTGCTTCCAAAAAG FCG14-rpGGGGACCACTTTGTACAAGAAAGCTGG 244 GTCCTAAGCTATTTTTTTAGAAACATT G FCG15-fpGGGGACAAGTTTGTACAAAAAAGCAGG 245 CTTGGTTCCGCGTGGTAGCATGAGACAAAAGCGTGCCAAG FCG15-rp GGGGACCACTTTGTACAAGAAAGCTGG 246GTCCTAGTTGTTGCTTCGTTCACTTGC FCG16-fp GGGGACAAGTTTGTACAAAAAAGCAGG 247CTTGGTTCCGCGTGGTAGCATGTCTGA AACAAAAAATATTG FCG16-rpGGGGACCACTTTGTACAAGAAAGCTGG 248 GTCCTACTTTCTTTTCTTTTTGGAAG FCG17-fpGGGGACAAGTTTGTACAAAAAAGCAGG 249 CTTGGTTCCGCGTGGTAGCATGAGGAT TTATCAATGTCAFCG17-rp GGGGACCACTTTGTACAAGAAAGCTGG 250 GTCCTAACACGTTTTTGTGTCACTTTC

Example 11 Biochemical Demonstration that CaYLR100w is a 3-Keto SterolReductase Involved in C-4 Sterol Demethylation

Overnight cultures were diluted into fresh SD +histidine (His) broth andgrown at 30° C. until logarithmic growth was established. Cultures wereharvested by centrifugation. Cells were then separated into tubes at afinal density of 0.25 OD₆₀₀ containing either fresh SD +His broth or SD+His broth containing 5 mM methionine (Met) and 2.5 mM cysteine (Cys).At the end of either a 0.75, 1.5, 3 or 4.5 hour incubation period, theOD₆₀₀ values of each tube were measured and cultures were adjusted to adensity of 0.25 OD₆₀₀ units per ml (2 mls per tube). Cultures were thenpulsed with 2 μCi/tube of [³H]-acetic acid for 60 minutes.

At the end of the labeling period, cells were pelleted and rinsed 1×with medium. The washed pellet was resuspended with 1 ml of 0.1 N HCl,transferred to glass screw-capped tubes, and incubated for 5 minutes at85° C. Two mls of 90% ethanol: 15% KOH: 0.25% pyrogallol (w/v) was addedand the sample allowed to saponify for 30 minutes at 85° C.

Tubes were extracted with three mls of petroleum ether with vigorousvortexing. The ether layer was removed and the residual aqueous layerwas extracted a second time with 3 mls of petroleum ether. The etherlayers were pooled and evaporated to dryness under an N₂ stream.

Dried samples were disolved in petroleum ether and spotted onto a 20cm×20 cm silica gel 60 TLC plate. The plate was developed in a solventsystem of benzene: ethyl acetate (99.5:0.5% v/v) until solvent front was2 cm from the top of the TLC plate. The plate was dried completely thenexposed for 1 to 3 days against X-ray film. The location of radiolabeledspots on the TLC plate was determined by matching the exposed spots onthe autoradiogram. Results are shown in FIGS. 22A and B.

The affect of downregulating CaYLR100w expression on cell growth wasalso assessed by tracking the absorbance at OD₆₀₀ for both thecaerg1Δ/P_(MET3-)CaERG1 (FIG. 23A) and fcg5Δ/P_(MET3-)FCG5 (FIG. 23B) inthe presence or absence of methionine and cysteine.

In order to quantitate the radioactivity associated with the ergosterolpathway products, the silica gel corresponding to the exposed spots wasexcised from the plate and mixed with 0.5 ml of water, then mixed with 5mls of scintillation cocktail prior to scintillation counting.[¹⁴C]-cholesterol was used as a standard for the migration of ergosterolon the TLC plate. Results are shown in FIG. 24.

Example 12 Biochemical Demonstration that CaYDR341c is Involved in WholeCell Protein Synthesis

A plate containing SD +His agar was inoculated with C. albicans mutantfcg6Δ/P_(MET3-)FCG6 and incubated for 2 days at 30° C. An overnightculture was prepared from several colonies in SD +His broth andincubated at 30° C. on a rotary wheel for 18 hours. The overnightculture was diluted to 0.125 OD₆₀₀ and incubated at 30° C. until 0.28OD₆₀₀ was achieved. A 100 μl aliquot of this culture was pipetted intothe wells of a 96-well filter plate. Half of the wells then received 50μl of SD +His broth, while the other half received 50 μl of SD +Hisbroth containg 15 mM Met and 7.5 mM Cys solution to give a finalconcentration of methionine and cysteine of 5 mM and 2.5 mM,respectively. The plate was incubated at 30° C. without shaking for 3.5hours prior to labelling.

A 4× radiolabelled amino acid labelling stock was prepared by adding 20μl of 1 mCi/ml [³H]-leucine (42.5 Ci/mmole) or [³H]-arginine (57Ci/mmole) to 1 ml of SD +His broth. Wells were labelled for 1 hour at30° C. with the addition of 50 μl of the above [³H]-leucine or[³H]-arginine solution into the well (Final radioactive concentration of5 μCi/ml or 1 μCi per well). After one hour, labelling was terminatedwith the addition of 100 μl of 20% trichloroacetic acid (TCA) per well(final [TCA]=6.7%). Plates were incubated at 4° C. overnight toprecipitate proteins and amino acid-charged tRNA. Precipitates werecollected by filtering on a vacumn manifold, washing filter wells 2×with 10% TCA, then 2× with water and finally 1× with ethanol. The platewas counted by adding 100 μl of Microscint-A scintillation fluid to thedried filter plate, then counting in a Packard Top-count scintillationcounter. Results are shown in FIGS. 25A and 25B.

Example 13 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in herein, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Ampr), a bacterial origin of replication (ori),an IPTG-regulatable promoter/operator (P/O), a ribosome binding site(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, that expresses the lacI repressorand also confers kanamycin resistance (Kanr). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased polynucleotide expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4 degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind tothe Ni-NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4 degree C. or frozen at −80 degree C.

Other methods of purifying a polypeptide of the present invention areknown in the art or disclosed elsewhere herein.

Example 14 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (Poros HQ-50, PerceptiveBiosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchangeresins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0.Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.The CM-20 column is then eluted using a 10 column volume linear gradientranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50mM sodium acetate, pH 6.5. Fractions are collected under constant A280monitoring of the effluent. Fractions containing the polypeptide(determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Coomassie blue stained 16% SDS-PAGE gel when 5ug of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Other methods of purifying a polypeptide of the present invention areknown in the art or disclosed elsewhere herein.

Example 15 Cloning and Expression of a Polypeptide in a BaculovirusExpression System

In this example, the plasmid shuttle vector pAc373 is used to insert apolynucleotide into a baculovirus to express a polypeptide. A typicalbaculovirus expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby convenient restriction sites, which may include, for example BamHI,Xba I and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is often used for efficient polyadenylation. For easy selectionof recombinant virus, the plasmid contains the beta-galactosidasepolynucleotide from E. coli under control of a weak Drosophila promoterin the same orientation, followed by the polyadenylation signal of thepolyhedrin polynucleotide. The inserted polynucleotides are flanked onboth sides by viral sequences for cell-mediated homologous recombinationwith wild-type viral DNA to generate a viable virus that express thecloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pVL941 and pAcIM1, as one skilled in the art would readilyappreciate, as long as the construct provides appropriately locatedsignals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 12, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites at the 5′ end of the primers inorder to clone the amplified product into the expression vector.Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified elsewhere herein (if applicable), is amplified usingthe PCR protocol described herein. If the naturally occurring signalsequence is used to produce the protein, the vector used does not need asecond signal peptide. Alternatively, the vector can be modified toinclude a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures,” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratapolynucleotide Cloning Systems, La Jolla, Calif.) cellsare transformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transformedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGoldtm baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldtm virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Other methods of purifying a polypeptide of the present invention areknown in the art or disclosed elsewhere herein.

Example 16 Expression of the Fungal Conserved Essential Polypeptides inMammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transformation with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transformedcells.

The transformed polynucleotide can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the polynucleotide of interest. (See,e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin,J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page,M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992). Using these markers, the mammalian cells are grown inselective medium and the cells with the highest resistance are selected.These cell lines contain the amplified gene(s) integrated into achromosome. Chinese hamster ovary (CHO) and NSO cells are often used forthe production of proteins.

A polynucleotide of the present invention is amplified according to theprotocol outlined in herein. If the naturally occurring signal sequenceis used to produce the protein, the vector does not need a second signalpeptide. Alternatively, if the naturally occurring signal sequence isnot used, the vector can be modified to include a heterologous signalsequence. (See, e.g., WO 96/34891.) The amplified fragment is isolatedfrom a 1% agarose gel using a commercially available kit (“Geneclean,”BIO 101 Inc., La Jolla, Calif.). The fragment then is digested withappropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR polynucleotide isused for transformation. Five pg of an expression plasmid iscotransformed with 0.5 ug of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo polynucleotide from Tn5 encoding an enzymethat confers resistance to a group of antibiotics including G418. Thecells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.After 2 days, the cells are trypsinized and seeded in hybridoma cloningplates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25,or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 dayssingle clones are trypsinized and then seeded in 6-well petri dishes or10 ml flasks using different concentrations of methotrexate (50 nM, 100nM, 200 nM, 400 nM, 800 nM). Clones growing at the highestconcentrations of methotrexate are then transferred to new 6-well platescontaining even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM,10 mM, 20 mM). The same procedure is repeated until clones are obtainedwhich grow at a concentration of 100-200 uM. Expression of the desiredpolynucleotide product is analyzed, for instance, by SDS-PAGE andWestern blot or by reversed phase HPLC analysis.

Other methods of purifying a polypeptide of the present invention areknown in the art or disclosed elsewhere herein.

Example 17 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention Through Molecular Evolution

Although many of the most biologically active proteins known are highlyeffective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, pharmaceutical, and/or industrial applications. Among thesetraits, a short physiological half-life is the most prominent problem,and is present either at the level of the protein, or the level of theproteins mRNA. The ability to extend the half-life, for example, wouldbe particularly important for a proteins use in polynucleotide therapy,transgenic animal production, the bioprocess production and purificationof the protein, and use of the protein as a chemical modulator amongothers. Therefore, there is a need to identify novel variants ofisolated proteins possessing characteristics which enhance theirapplication as a therapeutic for treating diseases of animal origin, inaddition to the proteins applicability to common industrial andpharmaceutical applications.

Thus, one aspect of the present invention relates to the ability toenhance specific characteristics of invention through directed molecularevolution. Such an enhancement may, in a non-limiting example, benefitthe inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

For example, an engineered CURF may be constitutively active uponbinding of its cognate ligand. Alternatively, an engineered CURF may beconstitutively active in the absence of ligand binding. In yet anotherexample, an engineered CURF may be capable of being activated with lessthan all of the regulatory factors and/or conditions typically requiredfor CURF activation (e.g., ligand binding, phosphorylation,conformational changes, etc.). Such CURFs would be useful in screens toidentify CURF modulators, among other uses described herein.

Directed evolution is comprised of several steps. The first step is toestablish a library of variants for the polynucleotide or protein ofinterest. The most important step is to then select for those variantsthat entail the activity you wish to identify. The design of the screenis essential since your screen should be selective enough to eliminatenon-useful variants, but not so stringent as to eliminate all variants.The last step is then to repeat the above steps using the best variantfrom the previous screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

Random mutagenesis has been the most widely recognized method to date.Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as descibed by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

While both of the aforementioned methods are effective for creatingrandomized pools of macromolecule variants, a third method, termed “DNAShuffling”, or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) hasrecently been elucidated. DNA shuffling has also been referred to as“directed molecular evolution”, “exon-shuffling”, “directed enzymeevolution”, “in vitro evolution”, and “artificial evolution”. Suchreference terms are known in the art and are encompassed by theinvention. This new, preferred, method apparently overcomes thelimitations of the previous methods in that it not only propagatespositive traits, but simultaneously eliminates negative traits in theresulting progeny.

DNA shuffling accomplishes this task by combining the principal of invitro recombination, along with the method of “error-prone” PCR. Ineffect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments-further diversifying the potential hybridation sitesduring the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNAshuffling reaction. However, specific reaction conditions for DNAshuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction.Preparation may be in the form of simply purifying the DNA fromcontaminating cellular material, chemicals, buffers, oligonucleotideprimers, deoxynucleotides, RNAs, etc., and may entail the use of DNApurification kits as those provided by Qiagen, Inc., or by the Promega,Corp., for example.

Once the DNA substrate has been purified, it would be subjected to DnaseI digestion. About 2-4 ug of the DNA substrate(s) would be digested with0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resultingfragments of 10-50 bp could then be purified by running them through a2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatman) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cuttoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCL,followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 nM Tris.HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60s; 94 C for 30s, 50-55 C for 30s, and 72 C for 30s using 30-45cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30s, 50 C for 30s, and72 C for 30s). The referred primers would be primers corresponding tothe nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purifiedusing methods well known in the art (e.g., Qiagen PCR purification kits)and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published todate, such variations would be obvious to the skilled artisan and areencompassed by the invention. The DNA shuffling method can also betailered to the desired level of mutagenesis using the methods describedby Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).

As described above, once the randomized pool has been created, it canthen be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficialmutations. When combined with screening, DNA shuffling allows thediscovery of the best mutational combinations and does not assume thatthe best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

Finally, recombination enables parallel processing. This represents asignificant advantage since there are likely multiple characteristicsthat would make a protein more desirable (e.g. solubility, activity,etc.). Since it is increasingly difficult to screen for more than onedesirable trait at a time, other methods of molecular evolution tend tobe inhibitory. However, using recombination, it would be possible tocombine the randomized fragments of the best representative variants forthe various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host, particularly if the polynucleotides andpolypeptides provide a therapeutic use. For example, a particularvariant of the present invention may be created and isolated using DNAshuffling technology. Such a variant may have all of the desiredcharacteristics, though may be highly immunogenic in a host due to itsnovel intrinsic structure. Specifically, the desired characteristic maycause the polypeptide to have a non-native structure which could nolonger be recognized as a “self” molecule, but rather as a “foreign”,and thus activate a host immune response directed against the novelvariant. Such a limitation can be overcome, for example, by including acopy of the polynucleotide sequence for a xenobiotic ortholog of thenative protein in with the polynucleotide sequence of the novel variantpolynucleotide in one or more cycles of DNA shuffling. The molar ratioof the ortholog and novel variant DNAs could be varied accordingly.Ideally, the resulting hybrid variant identified would contain at leastsome of the coding sequence which enabled the xenobiotic protein toevade the host immune system, and additionally, the coding sequence ofthe original novel varient that provided the desired characteristics.

Likewise, the invention encompasses the application of DNA shufflingtechnology to the evolution of polynucletotides and polypeptides of theinvention, wherein one or more cycles of DNA shuffling include, inaddition to the polynucleotide template DNA, oligonucleotides coding forknown allelic sequences, optimized codon sequences, known variantsequences, known polynucleotide polymorphism sequences, known orthologsequences, known homolog sequences, additional homologous sequences,additional non-homologous sequences, sequences from another species, andany number and combination of the above.

In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in polynucleotide therapy,protein engineering, evolution of whole cells containing the variant, orin the evolution of entire enzyme pathways containing polynucleotides ofthe invention as described in PCT applications WO 98/13485, WO 98/13487,WO 98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. The forgoing are hereby incorporated in their entiretyherein for all purposes.

Example 18 Method of Creating N- and C-Terminal Deletion MutantsCorresponding to the Fungal Essential Conserved Polypeptides of thePresent Invention

As described elsewhere herein, the present invention encompasses thecreation of N- and C-terminal deletion mutants, in addition to anycombination of N- and C-terminal deletions thereof, corresponding to thefungal essential conserved polypeptides of the present invention. Anumber of methods are available to one skilled in the art for creatingsuch mutants. Such methods may include a combination of PCRamplification and polynucleotide cloning methodology. Although one ofskill in the art of molecular biology, through the use of the teachingsprovided or referenced herein, and/or otherwise known in the art asstandard methods, could readily create each deletion mutant of thepresent invention, exemplary methods are described below.

Briefly, using the isolated cDNA clone encoding the full-length fungalessential conserved polypeptides sequence (as described in herein, forexample), appropriate primers of about 15-25 nucleotides derived fromthe desired 5′ and 3′ positions of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or 11 may be designed to PCR amplify, and subsequently clone, theintended N- and/or C-terminal deletion mutant. Such primers couldcomprise, for example, an initiation and stop codon for the 5′ and 3′primer, respectively. Such primers may also comprise restriction sitesto facilitate cloning of the deletion mutant post amplification.Moreover, the primers may comprise additional sequences, such as, forexample, flag-tag sequences, kozac sequences, or other sequencesdiscussed and/or referenced herein.

Representative PCR amplification conditions are provided below, althoughthe skilled artisan would appreciate that other conditions may berequired for efficient amplification. A 100 ul PCR reaction mixture maybe prepared using 10 ng of the template DNA (cDNA clone of fungalessential conserved polypeptides), 200 uM 4dNTPs, 1 uM primers, 0.25 UTaq DNA polymerase (PE), and standard Taq DNA polymerase buffer. TypicalPCR cycling condition are as follows:

20–25 cycles: 45 sec, 93 degrees  2 min, 50 degrees  2 min, 72 degrees 1cycle: 10 min, 72 degrees

After the final extension step of PCR, 5 U Klenow Fragment may be addedand incubated for 15 min at 30 degrees.

Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). The skilled artisan would appreciate that other plasmidscould be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E. coli cells usingmethods provided herein and/or otherwise known in the art.

The 5′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of the fungal essential conservedpolypeptides polynucleotide (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11), and ‘X’ is equal to the most N-terminal amino acid of theintended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 5′ primer, while the second termwill provide the end 3′ nucleotide position of the 5′ primercorresponding to sense strand of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or 11. Once the corresponding nucleotide positions of the primer aredetermined, the final nucleotide sequence may be created by the additionof applicable restriction site sequences to the 5′ end of the sequence,for example. As referenced herein, the addition of other sequences tothe 5′ primer may be desired in certain circumstances (e.g., kozacsequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:(S+(X*3)) to ((S+(X*3))-25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of the fungal essential conservedpolypeptides polynucleotide (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or 11), and ‘X’ is equal to the most C-terminal amino acid of theintended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 3′ primer, while the second termwill provide the end 3′ nucleotide position of the 3′ primercorresponding to the anti-sense strand of SEQ ID NO:1, 2, 3, 4, 5, 6, 7,8, 9, 10, or 11. Once the corresponding nucleotide positions of theprimer are determined, the final nucleotide sequence may be created bythe addition of applicable restriction site sequences to the 5′ end ofthe sequence, for example. As referenced herein, the addition of othersequences to the 3′ primer may be desired in certain circumstances(e.g., stop codon sequences, etc.). The skilled artisan would appreciatethat modifications of the above nucleotide positions may be necessaryfor optimizing PCR amplification.

The same general formulas provided above may be used in identifying the5′ and 3′ primer sequences for amplifying any C-terminal deletion mutantof the present invention. Moreover, the same general formulas providedabove may be used in identifying the 5′ and 3′ primer sequences foramplifying any combination of N-terminal and C-terminal deletion mutantof the present invention. The skilled artisan would appreciate thatmodifications of the above nucleotide positions may be necessary foroptimizing PCR amplification.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g.,polynucleotide therapy), agonists, and/or antagonists of polynucleotidesor polypeptides of the invention.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Further, the hard copy of the sequence listing submitted herewith andthe corresponding computer readable form are both incorporated herein byreference in their entireties.

Lengthy table referenced here US07465568-20081216-T00001 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US07465568-20081216-T00002 Please refer tothe end of the specification for access instructions.

Lengthy table referenced here US07465568-20081216-T00003 Please refer tothe end of the specification for access instructions.

LENGTHY TABLES The patent contains a lengthy table section. A copy ofthe table is available in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US07465568B2). Anelectronic copy of the table will also be available from the USPTO uponrequest and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. An isolated nucleic acid molecule consisting of a polynucleotidesequence selected from the group consisting of: (a) an isolatedpolynucleotide encoding a polypeptide consisting of amino acids 1 to 346of SEQ ID NO:12; and (b) an isolated polynucleotide encoding apolypeptide consisting of amino acids 2 to 346 of SEQ ID NO:12; whereinsaid encoded polypeptide has keto-steroid reductase activity.
 2. Theisolated nucleic acid molecule of claim 1, wherein said polynucleotideis (a).
 3. The isolated nucleic acid molecule of claim 2, wherein saidpolynucleotide consist of nucleotides 1 to 1038 of SEQ ID NO:1.
 4. Theisolated nucleic acid molecule of claim 1, wherein said polynucleotideis (b).
 5. The isolated nucleic acid molecule of claim 4, wherein saidpolynucleotide consist of nucleotides 4 to 1038 of SEQ ID NO:1.
 6. Arecombinant vector consisting of the isolated nucleic acid molecule ofclaim 1 operably linked to a promoter at the 5′ end and operably linkedto a stop codon at the 3′ end, wherein said vector comprises an originof replication and a selectable marker.
 7. An isolated recombinant hostcell comprising the vector sequence of claim
 6. 8. A method of making anisolated polypeptide comprising: (a) culturing the isolated recombinanthost cell of claim 7 under conditions such that said polypeptide isexpressed; and (b) recovering said polypeptide.
 9. An isolated nucleicacid molecule consisting of a polynucleotide having a nucleotidesequence that is at least 95.0% identical to a polynucleotide sequenceprovided in claim 1, wherein percent identity is calculated using aCLUSTALW global sequence alignment using default parameters, and whereinsaid polynucleotide encodes a polypeptide that has keto-steroidreductase activity.
 10. An isolated nucleic acid molecule consisting ofa polynucleotide encoding a polypeptide that is at least 95.0% identicalto amino acids 2 to 346 of SEQ ID NO:12, wherein percent identity iscalculated using a CLUSTALW global sequence alignment using defaultparameters, and wherein said polynucleotide encodes a polypeptide thathas keto-steroid reductase activity.
 11. An isolated polynucleotideencoding a polypeptide consisting of at least 318 contiguous amino acidsof SEQ ID NO:12, wherein said polypeptide has keto-steroid reductaseactivity.
 12. The isolated nucleic acid molecule of claim 11, whereinsaid polynucleotide consists of at least 954 contiguous nucleotides ofSEQ ID NO:1.
 13. An isolated polynucleotide consisting of the completecomplementary sequence of (a) or (b) of claim 1.